Chromatography (GC & hplc)
Short Description
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Description
ASSIGNMENT CHE 515 INSTUMENTAL CHEMISTR CHEMIS TRY Y HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) & GAS CHROMATOGRAPHY – MASS SPECTROSCOPY (GC-MS) PREPARED BY SITI NURAJJAR BINTI JAMI 201 !"2 "0! EH 220# PREPARED FOR DR $AMARIAH 2015
ABSTRACT
1%0GC-MS
1%1
INTRODUCTION Gas chromatography (GC) or gas-liquid chromatography (GLC) is on o! th typs o! gas chromatographic analysis" Th othr typ is gas-solid chromatography (G#C) $hich %asd on a solid stationary phas" Th rtntion o! analyts occurs %y th mans o! physical adsorption" &hil in GC' th partition o! analyt occurs %t$n a gasous mo%il phas and liquid stationary phas' on th sur!ac o! an inrt solid pacing or on th $alls o! th columns" In *+' ,artin and #yng suggstd th concpt o! GLC" Thy $r also rsponsi%l !or th liquid-liquid partition chromatography" This tchniqu $as usd as a tool in la%oratory practics" In *' th !irst GLC apparatus $as commrciali.d into th mart" Th application o! th GLC tchniqu has %n phnomnal" #inc thn' GLC tchnology has also gro$ and consquntly ld to many ad/ancd GC instrumnts in this modrn day" In gnral' th signi!icant componnts in GC instrumnt ar0 i) #tationary phas 1 ara o! disprsion o! analyts ii) ,o%il phas 1 liquid' supr!icial !luid' or in this cas gas that carry analyts to stationary phas iii) #ampl in2ction compartmnt 1 th port to introduc th sampl into th systm i/) Column 1 a d/ic to $hich th stationary phas is !i3d in" /) Dtctor 1 systm that idnti!is' rcords or indicats th /aria%ls changd in its n/ironmnt"
"4 T56OR7 8igur "9 sho$d a schmatic diagram o! th %asic componnt o! a typical Gas Chromatography Instrumntation" Th instrumnts in GC-,# systm ar %asically similar to any othr GC systm" &hat di!!r th GC-,# systm to th othr GC systm is th dtctor usd in a GC-,#" :s th nam proposd' a GC-,# systm must % connctd to th mass spctromtr to !unction as th dtctor o! th systm" Th instrumntation o! a GC-,# includs0 "4" Carrir gas systm0 In GC' th carrir gas systm consists o! th mo%il-phas gas' $hich mas it to % no$n as carrir gas" Th carrir gas usd in GC must % chmically inrt" In a common practic' hlium gas is usually usd" Dspit that' othr gass li argon' nitrogn and hydrogn ar also usd" Ths gass ar radily containd in prssuri.d tan" In ordr to control th !lo$ rats o! th carrir gas' th systm had to ha/ th prssur rgulator' gaugs and th !lo$ mtrs" Normally' th !lo$ rats ar controlld in t$o stags" Th !irst stag is at th gas cylindr' $hil th othr on is at th chromatograph" 8or pacd columns' th !lo$ rats usually st to th rang o! 4 1 9 mL;min' $hil !or th opn tu%ular capillary columns' th !lo$ rats ar usually st to 1 4 ml;min" In gnral' i! th inlt prssur rmains constant' th !lo$ rats ar assumd to rmain constant" Th inlt prssurs ar usually st to % at th rang o! 9 1 9 psi a%o/ room prssur" :s !or th gas !lo$ rats at th column' thy can % sta%lishd using a rotomtr at th column had" 5o$/r' a mor accurat d/ic to pr!orm this is th simpl soap-%u%%l mtr" &hil no$adays' as th tchnology ad/ancs' many gas chromatographs ar quippd $ith lctronic !lo$ mtrs $hich ar can % rgulatd through a computr to maintain th !lo$ "4"4
rat at th dsird l/l" #ampl in2ction systm0 Th sampl port' locatd at th had o! th column' nds to % prhatd %!or introducing th sampls into it" Typically' it is hatd to a tmpratur o! a%out 9 OC highr than th %oiling point o! th last /olatil componnt o! th sampl" #ampls ar in2ctd to th hatd sampl port through a ru%%r or silicon diaphragm or sptum" This is don %y using cali%ratd
microsyrings"
Th
sampl
is
usually
introducd
into
th
chromatographr in th !orm o! a L !or ordinary pacd analytical columns' $hil th capillary columns rquir sampls to % smallr %y a !actor o! 99 or mor" In most o! ad/ancd gas chromatographs' thy ar radily quippd $ith th :uto-in2ctors togthr $ith th automatic sampling trays" Ths impro/mnts in GC pro/id con/ninc !or sampl in2ction as thy hlp to o%tain a mor prcis /olum o! in2ctd sampls "4"?
compard to th manually sampl in2ction using th syring Column0 a) Column typs@ Thr ar t$o typs o! columns usd in GC0 pacd columns and opn tu%ular columns or also no$n as capillary columns" Opn tu%ular columns o!!r a highr !!icincy than th pacd columns" 5nc' capillary columns ar $idly usd no$adays" Opn Tu%ular Columns T$o typs o! opn tu%ular columns ar0 &all-coatd opn tu%ular (&COT) 1 capillary tu%s coatd $ith a thin layr o! stationary phas" #upport-coatd opn tu%ular (#COT) 1 th innr sur!ac o! th capillary is lind $ith a thin !ilm o! support matrial' diatomacous arth as such" #COT has a gratr sampl capacity than &COT' %ut in !!icincy aspct' &COT is mor !!icint than #COT" Th commonly usd capillary columns ar !usd-silica $all-coatd opn tu%ular columns (8#&C)' $hich ha/ thinnr $all and strngthn %y an outsid protcti/ polyimid coating" Ths columns also o!!r !l3i%ility in $hich thy ar a%l to %nd into coils $ith a !$ inchs o! diamtr" %) #tationary Ahas@ It is o!tn crucial to dcid $hich immo%ili.d liquid phas to % usd in th GC" #tationary phas can % chosn %asd on th proprtis o!0 lo$ing /olatility' thrmal sta%ility' chmical inrtnss
and sol/nt charactristics" Th guidlins to slct th %st stationary phas can % o%taind !rom litratur r/i$' intrnt sarch'
prior
3princ
or
ad/ic
!rom
th
/ndor
o!
chromatographic quipmnt and supplis" Th most $idly usd stationary phas ar polydimthyl silo3an' polythyln glycol and B Ahnyl-polydimtyl silo3an" Thir applications di!!r !rom ach othr' dpnding on th typs o! sampl analysd" c) Column con!iguration and column o/n tmpratur@ Th column is ordinarily housd $ith a thrmostattd o/n in ordr to control th tmpratur o! th column' sinc th column tmpratur in on o! th important /aria%ls to o%tain a prcis $or" asd on pr/ious studis' th tmpratur that st to % qual or a !$ dgrs a%o/ th a/rag %oiling point o! a sampl ld to an accpta%l rsult" Thus' th optimal tmpratur o! th column is st %asd on th %oiling point o! th sampl and th sparation dgr rquird" "4"+
Dtctor0 Thr ar many typs o! dtctor a/aila%l to % coupld as dtction systm to th GC" Thy dpnd on th typs o! analysis to % conductd" 63ampls o! th dtctors ar !lam ioni.ation dtctors' thrmal conducti/ity dtctors' lctron-captur dtctors' and atomic mission dtctors" &hil in a GC-,# systm' as th nam proposd' th mass spctromtr is coupld to th GC !or dtction purpos" 5nc' this systm a%l to hlp to idnti!y th analyts prsnt in a sampl' instad o! only dtcts thir apparanc" :nalysis %y mass spctromtr %gins $ith th !ormation o! gasous analyt ions" Th mass spctra rsults !rom a mass spctromtr dpnd on th mthod usd !or th ion !ormation" Th ion-sourcs commonly usd in GC;,# ar0 i) 6lctron-impact ioni.ation@ Th procsss occurrd in lctron impact ar0 a) #ampl is hatd to a tmpratur that is high nough to produc a molcular /apour %) Th molculs !ormd ar thn ioni.d %y %om%arding thm $ith a %am o! nrgtic lctrons"
Th nrgtic lctrons approach /ry closly to th molculs" This rsults in th loss o! lctrons !rom th molculs %y lctrostatic rpulsion" Th primary product o! th ion impact is singly chargd positi/ ions" Though th lctron-impact ioni.ation is lss !!icint' %ut th us o! this tchniqu is still important in many mass spctromtrs du to its con/nint' high ion currnts production and snsiti/ity" ii)
Chmical ioni.ation@ In chmical ioni.ation procss' ioni.ation o! gasous sampl atoms occurrd %y collision o! th atoms $ith ions producd %y lctron %om%ardmnt o! an 3css o! a ragnt gas" This procss also producs singly chargd positi/ ions" #om o! th ragnts usd in chmical ioni.ation ar mthan' propan' iso%utn and ammonia" Th us o! th ragnts rsults to a
di!!rnt spctrum according to analyts o! th analysis" ,odrn tchnology has ld to th dsignation o! mass spctromtr quippd $ith intrchanga%l ion sourc" Thus' it is a%l to do ithr lctron-impact ioni.ation or chmical impact ioni.ation" To pr!orm chmical impact ioni.ation' th lctron %am ioni.ation ara o! th lctron-impact ioni.ation is modi!id %y addition to th capacity o! /acuum pump and rduction o! th $idth o! th slit to mass analysr" Ths modi!ications lad lctron %ams to ract $ith ragnt molculs narly 3clusi/ly" 5nc' du to th addition o! proton in th prsnc o! th ragnt ions' th spctra !rom th chmical ioni.ation ar gnrally contain $ll-d!ind pas than lctronimpact ioni.ation spctra" In a nutshll' to run an analysis using a GC-,#' th sampl is !irstly in2ctd into th pr-hatd sampl port o! th systm' th carrir gas $ill !lo$ $ill carry th analyts o! th sampl to th column o! th gas chromatographic systm !or sparation' thn th output !rom th column is !d dirctly into th ioni.ation cham%r o! th mass spctromtr" 8inally' a schmatic illustration' $hich is th outcom o! th analysis' is printd out o! th output systm"
"? #:,AL6 AR6A:R:TION
"+ 6:,AL6 O8 :N:L7#I# :nalysis o! EOC contnt in th 3hald %rath o! to%acco cigartt and cigartt smors using GC-,C In a rsarch conductd %y 6sthr ,arco and Foan O" Grimalt' GC-,# $as usd to analys th /olatil organic compound contnt in th 3hald %rath o! to%acco cigartt and lctronic cigartt smors' $hich includd disposa%l -cigartt (Typ ) and rcharga%l -cigartt (Typ 4)" In thir analysis' sampl o! 3hald air $r collctd using a io-EOC 3hald %rath samplr" This d/ic $as d/lopd %y th U 5alth and #a!ty La%oratory" lo$ is th o/r/i$ o! th analysis tchniqus in/ol/d in sampl collction !or th analysis0 "+" #ampl Arparation Tchniqus@ i) !or asing /oluntrs to smo' thy $r asd to %lo$ at thir highst capacity into th io-EOC d/ic !irst through a disposa%l ii)
mouthpic" Eoluntrs $r asd to smo to%acco cigartt' %y inspird and 3pird
iii)
dply thr tims' thn rtain th %rath !or 49 sconds Eoluntrs %l$ at thir highst capacity into th io-EOC %ody through a
i/) /)
disposa%l card%oard mouthpic" #tp ii) $as rpatd !i/ tims" Th sampl !rom th io-EOC $as trans!rrd into a sor%nt cartridg through a scr$d-in plungr" Th EOCs o! to%acco cigartt 3hald
/i) /ii)
%rath $r accumulatd in th sam cartridg" #tps !rom ii) through i/) $r rpatd !or -cigartt Typ I and Typ II" #ampl o! am%int air %!or and a!tr smoing $r also collctd using th sam d/ic to compar it to th analysis o! 3hald %rath o! th /arious typs o! cigartt" "+"4
,thodology Componnts to % analysd@ nicotin' %n.n' tolun' naphthaln' propyln glycol' glycrin' and othr pollutants o! gnral concrn" #tandard solutions prpard !or sampl idnti!ication@ 4-mthyl%utan' -pntn' cis-4-pntn' trans-4-pntn' +-mthyl--pntn and nin standard solutions o! organics in mthanol $ith di!!rnt concntration ranging !rom 9">g;L to 499>g;L" Th opration o! th GC-,# systm0 ,odl@
Carrir gas@ hlium gas' controlld at "mL;min GC o/n tmpratur $as hold at +9oC !or th !irst 9 minuts' incrasd oC !or /ry minut until it rachd 9 oC" :!tr that' th tmpratur $as incrasd oC !or /ry minut until th tmpratur rachd 49oC" This Tmpratur $as hold !or 9 minuts" &hil th tmpratur o! th trans!r lin %t$n GC and ,# $as st to 4H9 oC" Columns@ Capillary column $ith 9m lngth' 9"?4mm intrnal diamtr' and >m !ilm thicnss 63primntal Arocdurs@ i) Th sampl port (thrmal dsor%r) $as pr-hatd to a ii)
tmpratur o! ?99oC" #tandard solutions $r prpard !rom commrcial solutions'
iii)
and thn introducd into clan sor%nt cartridgs" Th standard solutions $r thn analysd using th GC-,# to o%tain cali%ration cur/s' %y using introducing thm to th thrmal dsorption instrumnt (th sampl port) $hich
i/)
quippd $ith an auto-samplr" Thn' th analysis o! th EOCs !ollo$d" Th EOCs sampls $r trans!rrd !rom th cartridgs to th thrmal dsorption
/) /i)
instrumnt" Thn th systm $as run !or analysis" Th chromatographic pas illustrations $r printd out" Idnti!ication o! chromatographic pas o! th compounds $as %asd on th rtntion tim and r!rncs to th litratur o! mass spctrum
"+"?
Rsults 8igur and 8igur 4 sho$s th rsults !rom th analysis o! EOC contnt in th 3hald %rath o! to%acco cigartt and cigartt smors" Ths data could ld to a clu o! $hat compounds rtain in th rspiratory systm !rom smoing $hthr to%acco cigartt or -cigartt" Th am%int air o! th room $as compard %t$n th n/ironmnt %!or smoing and a!tr smoing in 8igur " :ccording to th discussion o! th rsults o! this 3primnt' th 3hald %rath n/ironmnt $as rich in acton and isoprn" Ths ar th typical compounds that can % idnti!id in this ind o! sampl" :s !or 8igur 4' th chromatograms o! th 3hald %rath o! th thr sampls $r compard to th sampl o! th n/ironmntal air o! th room a!tr smoing" Th chromatogram o! th 3hald air !rom to%acco cigartt sho$d a mor simpli!id mi3tur o! compounds' %sids lo$ring in th a%undanc o! som compound as compard to th smo chromatogram" This indicats that most o! th compounds o! th smo $r rtaind in th rspiratory systm" &hil !or th cigartt 3hald %rath chromatograms' %oth Typ and Typ 4 sho$d th a%snc o! propyln glycol and glycrin pas' $hich indicats that ths compounds $r rtaind in th lungs" sids that' th chromatogram o! th to%acco cigartt 3hald %rath sho$d a signi!icant di!!rnc to th chromatograms o!
th
-cigartt 3hald
%rath"
On
th
othr
hand'
th
chromatograms o! %oth Typ and Typ 4 -cigartt 3hald %rath $r almost similar" Comparing to%acco cigartt and -cigartt 3hald %raths' to%acco cigartt chromatograms illustratd a highr %urdn o! EOCs than -cigartt"
LIMITATIONS
HPLC INTRODUCTION 5igh-pr!ormanc liquid chromatography' 5ALC is a trm that can % usd intrchanga%ly $ith LC' liquid chromatography" Th trm $as usd to distinguish th mthod it rprsnts $ith th classic gra/ity-!lo$ liquid chromatography mthod" Th gnral instrumntation componnts consistd in LC is %asically similar to th GC' %ut thy di!!r in th typs o! th componnts usd' $hich $ill latr % 3plaind" Liquid chromatography historically %gan $ith th us o! glass column $ith diamtrs o! 9 1 9mm" Th stationary phas consistd o! solid particls' pacd $ith 9 1 99cm lngth" Th particls $r coatd $ith an adsor%d liquid" During that tim' s/ral modi!ications to th si. o! th particls $r mad to sta%lish a rasona%l !lo$ rats ho$/r !aild' %caus thy ld to longr tim consumption" Thn' attmpts to spd up th partition procss $as also mad %y putting on prssurs' %ut ld th incras o! th plat hight' $hich $as in!!icint" Liquid chromatography startd to d/lop $hn scintists rali.d that th pr!ormanc o! th column can % %oostd %y dcrasing th si. o! th particls o! pacings" Thn' in lat *9s' th tchnology to produc pacings $ith particls o! diamtr o! ? 1 9>m $as d/lopd" Th instrumntation o! LC %cam mor sophisticatd in contrast to th traditional gra/ity-!lo$ liquid chromatography' as th d/lopmnt o! th pacing particls rquird th opration o! th systm to % pr!ormd at high prssurs" LC is popularly usd in analytical sparation tchniqus du to its ad/antags" Ths ad/antags includ0 highr snsiti/ity' adapta%l to accurat quantitati/ dtrminations' asy to handl and suita%l !or sparation o! non-/olatil spcis" On top o! thos' th $idsprad o! th instrumnt is applica%l to su%stancs that ar important in th industry' many !ilds o! scinc and th pu%lic" Th su%stancs includ0 hydrocar%on' drugs' protins' car%ohydrats' psticids' stroids' anti%itics and /arious inorganic su%stancs"
T56OR7 Th typical componnts o! instrumnts in LC ar sho$n in 8igur " In modrn LC' prssurs ar pumpd at s/ral hundrd atmosphrs" This is rquird in ordr to achi/ rasona%l !lo$ rats !or pacing o! ? 1 9>m" 5nc' th 5ALC quipmnt is mor compl3 and costly compard to othr typs o! chromatographic instrumnts" Th instrumntations o! a gnral 5ALC ar la%oratd %lo$" ,o%il phas rsr/oirs and sol/nt tratmnt systm@ In a modrn LC apparatus' it may consist o! singl or multipl glass rsr/oirs" Th rsr/oirs ar containd $ith 99mL or mor liquid sol/nts" Arsnc o! dissol/d gass and dust !rom liquid sol/nts could intr!r th !lo$ rats and %and sprading' as $ll as th dtctor pr!ormanc" Thus' som rsr/oirs ha/ a %uilt in sparging systm' as sho$n in th !igur" Th spargr !unctions as dissol/d gasss rmo/r %y th mans o! !in %u%%ls o! inrt gas that is insolu%l in mo%il phas" Othr systms that may % %uilt in th dgassr ar /acuum pumping systm' a distillation systm' a d/ic !or hating and stirring' or as sho$n in th !igur' th inlt !iltr" It !unctions as a !iltration to th dust and particulat mattr in th sol/nts $hich can caus damag to th pumping systm and in2ction systm' and could also clog th column" In addition' partitioning /al/s ar also quippd into a modrn LC instrumnts to hlp th partitioning o! th sol/nt introducd in 5ALC at ratios that can % /arid continuously" Aumping systm@ Aumps ar rquird in LC to0 i) ii) iii) i/) /)
Gnrat prssurs to up to 999psi Aroduc puls-!r output Rgulat !lo$ rats at th rang o! 9" to "9 mL;min : %ttr !lo$ rproduci%ility Control corrosion %y sol/nts"
Though pump usd to incras th prssurs in LC' %ut ths high prssur liquids do not caus any ha.ardous 3plosion' sinc liquids ar not /ry comprssi%l" Th rsults o! ths high prssurs may only lad to componnt ruptur' and consquntly caus sol/nt laag" Instad' this laag may constitut to a !ir and may % ha.ardous to th n/ironmnt"
Th $idly usd pumps typs in LC ar th scr$-dri/n syring and th rciprocating pump" In most commrcial LC instrumnts' thy ar quippd $ith a computri.d systm in $hich thy ar mor con/nint in controlling th masurmnt o! th !lo$ rat" #ampl-in2ction systm #ampl introducd into th column pacing is rquird to % at a /ry small /olum" Th /olum rang may % o! a !$ tnths o! microlitr to o! a%out 99>L" This hlps to rduc th limiting !actor in LC prcision' $hich is th rproduci%ility $ith $hich sampl can % introducd into th column" Th common consr/ati/ $ay to introduc th sampl into LC is %y using sampling loops" Thy ar intgratd as a part o! th LC instrumnt and th loops can also % intrchanga%l dpnding on th si.s o! th sampl" Ths loops pro/id choics !or sampl si.s ranging !rom >L to 99>L" On th othr hand' most LC instrumnts in this modrn day ar quippd $ith auto-in2ctors systm" Th systm has sampling loops and syring pump $hich allo$ in2ction /olums to % ranging !rom lss than >L to mor than mL" &hil som systms allo$ !or controlling o! th n/ironmnt tmpratur o! th sampling port and som may % programma%l to allo$ !or unattndd in2ctions" Columns@ Thr ar s/ral typs o! LC columns in/ntd" Ths columns di!!r in trms o! physical constructions' si.s' pacing and costs" Usually' thy ar %uilt !rom smooth-%or stainlss stl tu%ing' somtims ha/y-$alld glass tu%ing and polymr tu%ing' and thy /n %uilt !rom stainlss stl' lind $ith glass or polymr" Common typs o! columns ar0 analytical columns and guard columns" Column tmpratur control@ #om applications o! LC do not rquir a clos control o! tmpratur" Thy opratd at room tmpratur" ut tmpratur rgulation is o!tn ncssary to o%tain %ttr chromatograms" ,ost ad/ancd instrumnts no$ ar quippd $ith hatrs to rgulat th tmpratur o! th column" sids that' som columns may also % !ittd $ith $atr 2act' $hich !unctioning as th tmpratur rgulator" Column pacing@
In 5ALC' thr ar t$o $ll-no$n typs o! column pacing usd0 Allicular particl 1 sphrical' nonporous' glass or polymr %ads $ith typical diamtrs o! ?9 1 +9>m" Thr $as a thin' porous layr dpositd on th sur!ac o! th %ads" This layr may % mad !rom silica' alumina' or an ion-3chang rsin" Aorous particl 1 consists o! porous microparticls $ith diamtrs ranging %t$n ? 1 9>m" Th particls ar mad !rom alumina' ion-3chang rsin' or th most commonly usd0 silica" Th silica particl o!tn coatd $ith thin organic !ilms" Th !ilms ar chmically or physically %ondd to th sur!ac" Dtctors Th idal proprtis o! dtctor usd in LC includ0 i) ii) iii) i/) /) /i)
:dquat snsiti/ity Good sta%ility and rproduci%ility Linar rspons to soluts that 3tnds o/r s/ral ordrs o! magnitud #hort rspons tim indpndnt o! !lo$ rat ,inimal intrnal /olum to rduc .on %roadning Compati%l $ith liquid !lo$"
Th dtctors commonly usd in 5ALC ar0 UE-Eisi%l a%sorption dtctors' !luorscnc dtctors' r!racti/-ind3 dtctors' and mass spctromtric dtctors" In gnral' th analysis run through th 5ALC is quit similar to th analysis %y GC systm" Th sampl is !irstly sampl port o! th systm' th mo%il phas $ill carry th analyts o! th sampl to th column o! th 5ALC systm !or sparation' thn th output !rom th column is !d into th dtctor d/ic" 8inally' a schmatic illustration' $hich is th outcom o! th analysis' is printd out o! th output systm"
#:,AL6 AR6A:R:TION : rsarch o! idnti!ication and quanti!ication o! %ioacti/ compounds in co!! %r$s $as conductd %y Naira Aornr Rodrigus and Nura ragagnolo" 5igh Ar!ormanc Liquid Chromatography (5ALC) $as in/ol/d in th mthodology o! th analysis" Thr $r s/ral typs o! sampls to % idnti!id and quanti!id in th analysis" Th sampls $r acquird !rom th local mart in th city o! Campina' #an Aaulo' ra.il" Th sampls $r also producd o! di!!rnt manu!acturrs" :s dscri%d in thir pacaging' th sampls consistd o! di!!rnt dgr o! idntity and quality' $hich mad thm to % classi!id into gourmt' traditional and suprm" sids that' thir dgr o! roasting also rangd %t$n light to dar" Th typs o! th sampls includ0 J rgular roastd ground co!!s (mard as RR)' ? dca!!inatd roastd ground co!!s (DR)' 4 rgular solu%l co!!s (R#)' and 4 dca!!inatd solu%l co!!s (D#)" Th roastd ground co!!s and th solu%l co!!s $r %r$d and prpard %y th ratio o! co!! to $atr as rcommndd in th pacaging" #ampl Arparation Tchniqus@ i) ii)
iii) i/) /)
To prpar th roastd ground co!!s %r$' g o! th sampls $r !irstly $ightd in a !iltr papr &hatman no" + 9 mL o! ultrapur $atr o! a%out *4 oC $as slo$ly pourd through th cntr o! th !iltrpapr" :nd thn' th solution $as trans!rrd into /olumtric !lass" Th !inal /olum o! th %r$ o! roastd ground co!!s /arid !rom ?+-?JmL" To prpar th solu%l co!!s %r$' 9"4g o! th sampls $r $ighd' and trans!rrd into 9mL /olumtric !lass" Thy $r dissol/d %y ultrapur $atr at tmpratur o! 4oC up to th cali%ration mars o! th !lass" Th %r$s o! th sampls $r immdiatly !ro.n %y liquid nitrogn" :nd thn' thy $r stord in a !r.r at th tmpratur o! -H9oC until analysis"
6:,AL6 O8 :N:L7#I# :nalysis o! %ioacti/ compounds in co!! %r$s %y 5ALC Th sampl prparation mthod $as listd in th sampl prparation part ' sction %rapa point %rapa" ,6T5ODOLOG7 Th analysis procdurs $r quit compl3' thus' this part is only !ocusing on th mthodology on using th 5ALC" Lin to th !ull /rsion o! th articl can % !ollo$d in th r!rncs" Componnts to % analysd@ ca!!in' chlorognic acids (CG:) and dri/ati/s' trigonllin' nicotinic acids' -hydro3ymthyl!ur!ural' tho%romin' thophyllin" #tandard solutions prpard !or sampl idnti!ication@ standards o! ca!!in (**B purity)' ca!!oylquinic acid (*B purity)' ca!!ic acid (*HB purity)' p-coumaric acid (*HB purity)' !rulic acid (*HB purity)' trigonllin hydrochlorid (*HB purity)' nicotinic acid (**"B purity)' -hydro3ymthyl!ur!ural (**B purity)' tho%romin (**B purity)' thophyllin (**B purity)" Th opration o! th 5ALC systm0 ,odl@ #himad.u 5ALC' quippd $ith %inary pump' dgassr $ith hlium' and automatic in2ction systm" 8or quantitati/ analysis' th 5ALC systm $as coupld to a diod array dtctor (D:D)' $hil !or compound idnti!ication purpos' it $as coupld $ith th mass spctromtr (,#)" ,o%il phas@ mi3tur o! H9B (/;/) 9m, citric acid (p5 4)' 49B (/;/) mthanol and 99B (/;/) mthanol !or ca!!in and CG: and its dri/ati/s analysis" &hil !or th othr compounds analysis' th mo%il phas includs0 9"B (/;/) actic acid (p5 ?) and mthanol" Columns@ 8or ca!!in and CG: and its dri/ati/ analysis' OD#-CH #him-pac column o! m' 4+9mm +"mm innr diamtr $as usd" &hil th othr analysis' th sam typ o! column $as also usd" 63primntal Arocdurs@
i)
Th co!! %r$s $r tratd !irstly to prcipitat th compounds o! highr molcular
ii)
$ight
%y
using
.inc
actat
solution
and
potassium
h3acyano!rrat solution" 8or ca!!in' and CG: and its dri/ati/s dtrmination' mL o! ach co!! %r$s $r trans!rrd into tst tu%s' and thn addd $ith 4mL o! ultrapur $atr" N3t' 9"mL o! .inc actat solution and potassium h3acyano!rrat
iii)
solution' and 9"HmL o! mthanol $r addd into th %r$ solution" Th mi3turs $r shan !or ?9 sconds' and thn l!t to sttl !or 9
i/)
minuts" Th prcipitat $as sparatd !rom th mi3turs %y cntri!ugation" Th
/)
suprnatants $r collctd' and thn !iltrd using 9"+>m mm%ran !iltrs" Th analysis $as carrid out using 5ALC-D:D and 5ALC-D:D-,#" !or analysing th sampls' standard solutions $r analysd in th systms %y introducing thm to th auto-samplr o! th systm" Thn' analysis o! th
/i)
sampls !ollo$d" &hil !or th othr compounds dtrmination' th %r$s $r tratd using
/ii) /iii)
9"4mL o! aquous lad actat solution" Th analysis using 5ALC systms !ollo$d" Th mass spctra illustrations $r printd out and discussd quantitati/' and qualitati/ly %y comparing th spctra to th litratur"
R6#ULT# Idnti!ication o! ca!!in and CG: and dri/ati/s@
Idnti!ication o! trigonllin' nicotinic acids' -5,8' tho%romin and thophyllin"
8igur sho$s th chromatogram !or th analysis o! ca!!in and CG: and dri/ati/s" asd on $hat had %n rportd in th analysis' %sids ca!!in' thr $r 4 dri/ati/s o! hydro3ycinnamic" Thy $r classi!id into + groups0 CG: (pas ' 4' +' ' J' ' 4' H' *' 49' 4' 44' 4?' 4' 4)' chlorognic acid lactons (pas 9' ?' ' J)' cinnamoyl-amino acid con2ugats (pas 4+' 4J) and !r cinnamic acid (pa H)" y comparison to th UE-/isi%l and mass spctra o! authntic standards' th compounds o! trigonllin' nicotinic acids' -5,8' tho%romin and thophyllin $r idnti!id in th chromatogram in 8igur 4" Th quanti!ication $as also don to !ind th amount o! %ioacti/ compounds contnt in th co!! %r$s' and th rsults $r summari.d as in Ta%l "
LIMITATIONSREFERENCES ''**+,%./-,%,3*S00214"!1501021*1-/2%0-S00214"!15010213+6%789'6:100!1-75-115-4+100000++;0702&+,+':1##00#4#091!;5!4!1#040257#+41!#50;# .,2- ''**+,%./,%,3%%?6'3%?%3>*S0415!510011""*1-/2%0S0415!510011""-3+6%789'6:++2;"-4027-115-+;700000++,;"1&+,+':1##04!149,2@+!,20;4"##07++!,+;""2!
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