Chromatography (GC & hplc)

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 typs o! gas chromatographic analysis" Th othr typ is gas-solid chromatography (G#C) $hich %asd on a solid stationary phas" Th rtntion o! analyts occurs %y th mans o! physical adsorption" &hil in GC' th partition o! analyt occurs %t$n a gasous mo%il phas and liquid stationary phas' on th sur!ac o! an inrt solid pacing or on th $alls o! th columns" In *+' ,artin and #yng suggstd th concpt o! GLC" Thy $r also rsponsi%l !or th liquid-liquid partition chromatography" This tchniqu $as usd as a tool in la%oratory practics" In *' th !irst GLC apparatus $as commrciali.d into th mart" Th application o! th GLC tchniqu has %n  phnomnal" #inc thn' GLC tchnology has also gro$ and consquntly ld to many ad/ancd GC instrumnts in this modrn day" In gnral' th signi!icant componnts in GC instrumnt ar0 i) #tationary phas 1 ara o! disprsion o! analyts ii) ,o%il phas 1 liquid' supr!icial !luid' or in this cas gas that carry analyts to stationary phas iii) #ampl in2ction compartmnt 1 th port to introduc th sampl into th systm i/) Column 1 a d/ic to $hich th stationary phas is !i3d in" /) Dtctor 1 systm that idnti!is' rcords or indicats th /aria%ls changd in its n/ironmnt"

"4 T56OR7 8igur "9 sho$d a schmatic diagram o! th %asic componnt o! a typical Gas Chromatography Instrumntation" Th instrumnts in GC-,# systm ar %asically similar to any othr GC systm" &hat di!!r th GC-,# systm to th othr GC systm is th dtctor usd in a GC-,#" :s th nam proposd' a GC-,# systm must % connctd to th mass spctromtr to !unction as th dtctor o! th systm" Th instrumntation o! a GC-,# includs0 "4" Carrir gas systm0 In GC' th carrir gas systm consists o! th mo%il-phas gas' $hich mas it to % no$n as carrir gas" Th carrir gas usd in GC must  % chmically inrt" In a common practic' hlium gas is usually usd" Dspit that' othr gass li argon' nitrogn and hydrogn ar also usd" Ths gass ar radily containd in prssuri.d tan" In ordr to control th !lo$ rats o! th carrir gas' th systm had to ha/ th prssur rgulator' gaugs and th !lo$ mtrs" Normally' th !lo$ rats ar controlld in t$o stags" Th !irst stag is at th gas cylindr' $hil th othr on is at th chromatograph" 8or pacd columns' th !lo$ rats usually st to th rang o! 4 1 9 mL;min' $hil !or th opn tu%ular capillary columns' th !lo$ rats ar usually st to  1 4 ml;min" In gnral' i! th inlt prssur rmains constant' th !lo$ rats ar assumd to rmain constant" Th inlt prssurs ar usually st to % at th rang o! 9 1 9 psi a%o/ room prssur" :s !or th gas !lo$ rats at th column' thy can % sta%lishd using a rotomtr at th column had" 5o$/r' a mor accurat d/ic to pr!orm this is th simpl soap-%u%%l mtr" &hil no$adays' as th tchnology ad/ancs' many gas chromatographs ar quippd $ith lctronic !lo$ mtrs $hich ar can % rgulatd through a computr to maintain th !lo$ "4"4

rat at th dsird l/l" #ampl in2ction systm0 Th sampl port' locatd at th had o! th column' nds to % prhatd %!or introducing th sampls into it" Typically' it is hatd to a tmpratur o! a%out 9 OC highr than th %oiling point o! th last /olatil componnt o! th sampl" #ampls ar in2ctd to th hatd sampl port through a ru%%r or  silicon diaphragm or sptum" This is don %y using cali%ratd

microsyrings"

Th

sampl

is

usually

introducd

into

th

chromatographr in th !orm o! a L !or ordinary pacd analytical columns' $hil th capillary columns rquir sampls to % smallr %y a !actor o! 99 or mor" In most o! ad/ancd gas chromatographs' thy ar radily quippd $ith th :uto-in2ctors togthr $ith th automatic sampling trays" Ths impro/mnts in GC pro/id con/ninc !or sampl in2ction as thy hlp to o%tain a mor prcis /olum o! in2ctd sampls "4"?

compard to th manually sampl in2ction using th syring Column0 a) Column typs@ Thr ar t$o typs o! columns usd in GC0 pacd columns and opn tu%ular columns or also no$n as capillary columns" Opn tu%ular columns o!!r a highr !!icincy than th pacd columns" 5nc' capillary columns ar $idly usd no$adays" Opn Tu%ular Columns T$o typs o! opn tu%ular columns ar0 &all-coatd opn tu%ular (&COT) 1 capillary tu%s coatd $ith a thin layr o! stationary phas" #upport-coatd opn tu%ular (#COT) 1 th innr sur!ac o! th capillary is lind $ith a thin !ilm o! support matrial' diatomacous arth as such" #COT has a gratr sampl capacity than &COT' %ut in !!icincy aspct' &COT is mor !!icint than #COT" Th commonly usd capillary columns ar !usd-silica $all-coatd opn tu%ular  columns (8#&C)' $hich ha/ thinnr $all and strngthn %y an outsid protcti/ polyimid coating" Ths columns also o!!r  !l3i%ility in $hich thy ar a%l to %nd into coils $ith a !$ inchs o! diamtr"  %) #tationary Ahas@ It is o!tn crucial to dcid $hich immo%ili.d liquid phas to % usd in th GC" #tationary phas can % chosn %asd on th  proprtis o!0 lo$ing /olatility' thrmal sta%ility' chmical inrtnss

and sol/nt charactristics" Th guidlins to slct th %st stationary phas can % o%taind !rom litratur r/i$' intrnt sarch'

prior

3princ

or

ad/ic

!rom

th

/ndor

o! 

chromatographic quipmnt and supplis" Th most $idly usd stationary phas ar polydimthyl silo3an' polythyln glycol and B Ahnyl-polydimtyl silo3an" Thir applications di!!r  !rom ach othr' dpnding on th typs o! sampl analysd" c) Column con!iguration and column o/n tmpratur@ Th column is ordinarily housd $ith a thrmostattd o/n in ordr  to control th tmpratur o! th column' sinc th column tmpratur in on o! th important /aria%ls to o%tain a prcis $or" asd on pr/ious studis' th tmpratur that st to % qual or a !$ dgrs a%o/ th a/rag %oiling point o! a sampl ld to an accpta%l rsult" Thus' th optimal tmpratur o! th column is st %asd on th %oiling point o! th sampl and th sparation dgr rquird" "4"+

Dtctor0 Thr ar many typs o! dtctor a/aila%l to % coupld as dtction systm to th GC" Thy dpnd on th typs o! analysis to % conductd" 63ampls o! th dtctors ar !lam ioni.ation dtctors' thrmal conducti/ity dtctors' lctron-captur dtctors' and atomic mission dtctors" &hil in a GC-,# systm' as th nam proposd' th mass spctromtr is coupld to th GC !or dtction purpos" 5nc' this systm a%l to hlp to idnti!y th analyts prsnt in a sampl' instad o! only dtcts thir apparanc" :nalysis %y mass spctromtr %gins $ith th !ormation o! gasous analyt ions" Th mass spctra rsults !rom a mass spctromtr  dpnd on th mthod usd !or th ion !ormation" Th ion-sourcs commonly usd in GC;,# ar0 i) 6lctron-impact ioni.ation@ Th procsss occurrd in lctron impact ar0 a) #ampl is hatd to a tmpratur that is high nough to  produc a molcular /apour   %) Th molculs !ormd ar thn ioni.d %y %om%arding thm $ith a %am o! nrgtic lctrons"

Th nrgtic lctrons approach /ry closly to th molculs" This rsults in th loss o! lctrons !rom th molculs %y lctrostatic rpulsion" Th primary product o! th ion impact is singly chargd positi/ ions" Though th lctron-impact ioni.ation is lss !!icint' %ut th us o! this tchniqu is still important in many mass spctromtrs du to its con/nint' high ion currnts  production and snsiti/ity" ii)

Chmical ioni.ation@ In chmical ioni.ation procss' ioni.ation o! gasous sampl atoms occurrd %y collision o! th atoms $ith ions producd %y lctron %om%ardmnt o! an 3css o! a ragnt gas" This  procss also producs singly chargd positi/ ions" #om o! th ragnts usd in chmical ioni.ation ar mthan' propan' iso%utn and ammonia" Th us o! th ragnts rsults to a

di!!rnt spctrum according to analyts o! th analysis" ,odrn tchnology has ld to th dsignation o! mass spctromtr  quippd $ith intrchanga%l ion sourc" Thus' it is a%l to do ithr  lctron-impact ioni.ation or chmical impact ioni.ation" To pr!orm chmical impact ioni.ation' th lctron %am ioni.ation ara o! th lctron-impact ioni.ation is modi!id %y addition to th capacity o!  /acuum pump and rduction o! th $idth o! th slit to mass analysr" Ths modi!ications lad lctron %ams to ract $ith ragnt molculs narly 3clusi/ly" 5nc' du to th addition o! proton in th prsnc o! th ragnt ions' th spctra !rom th chmical ioni.ation ar gnrally contain $ll-d!ind pas than lctronimpact ioni.ation spctra" In a nutshll' to run an analysis using a GC-,#' th sampl is !irstly in2ctd into th pr-hatd sampl port o! th systm' th carrir gas $ill !lo$ $ill carry th analyts o! th sampl to th column o! th gas chromatographic systm !or sparation' thn th output !rom th column is !d dirctly into th ioni.ation cham%r o! th mass spctromtr" 8inally' a schmatic illustration' $hich is th outcom o! th analysis' is printd out o! th output systm"

"? #:,AL6 AR6A:R:TION

"+ 6:,AL6 O8 :N:L7#I# :nalysis o! EOC contnt in th 3hald %rath o! to%acco cigartt and cigartt smors using GC-,C In a rsarch conductd %y 6sthr ,arco and Foan O" Grimalt' GC-,# $as usd to analys th /olatil organic compound contnt in th 3hald %rath o! to%acco cigartt and lctronic cigartt smors' $hich includd disposa%l -cigartt (Typ ) and rcharga%l -cigartt (Typ 4)" In thir analysis' sampl o!  3hald air $r collctd using a io-EOC 3hald %rath samplr" This d/ic $as d/lopd %y th U 5alth and #a!ty La%oratory" lo$ is th o/r/i$ o!  th analysis tchniqus in/ol/d in sampl collction !or th analysis0 "+" #ampl Arparation Tchniqus@ i) !or asing /oluntrs to smo' thy $r asd to %lo$ at thir  highst capacity into th io-EOC d/ic !irst through a disposa%l ii)

mouthpic" Eoluntrs $r asd to smo to%acco cigartt' %y inspird and 3pird

iii)

dply thr tims' thn rtain th %rath !or 49 sconds Eoluntrs %l$ at thir highst capacity into th io-EOC %ody through a

i/) /)

disposa%l card%oard mouthpic" #tp ii) $as rpatd !i/ tims" Th sampl !rom th io-EOC $as trans!rrd into a sor%nt cartridg through a scr$d-in plungr" Th EOCs o! to%acco cigartt 3hald

/i) /ii)

 %rath $r accumulatd in th sam cartridg" #tps !rom ii) through i/) $r rpatd !or -cigartt Typ I and Typ II" #ampl o! am%int air %!or and a!tr smoing $r also collctd using th sam d/ic to compar it to th analysis o! 3hald %rath o! th /arious typs o! cigartt" "+"4

,thodology Componnts to % analysd@ nicotin' %n.n' tolun' naphthaln'  propyln glycol' glycrin' and othr pollutants o! gnral concrn" #tandard solutions prpard !or sampl idnti!ication@ 4-mthyl%utan' -pntn' cis-4-pntn' trans-4-pntn' +-mthyl--pntn and nin standard solutions o! organics in mthanol $ith di!!rnt concntration ranging !rom 9">g;L to 499>g;L" Th opration o! th GC-,# systm0 ,odl@

Carrir gas@ hlium gas' controlld at "mL;min GC o/n tmpratur $as hold at +9oC !or th !irst 9 minuts' incrasd oC !or /ry minut until it rachd 9 oC" :!tr that' th tmpratur $as incrasd oC !or /ry minut until th tmpratur rachd 49oC" This Tmpratur $as hold !or 9 minuts" &hil th tmpratur o! th trans!r lin %t$n GC and ,# $as st to 4H9 oC" Columns@ Capillary column $ith 9m lngth' 9"?4mm intrnal diamtr' and >m !ilm thicnss 63primntal Arocdurs@ i) Th sampl port (thrmal dsor%r) $as pr-hatd to a ii)

tmpratur o! ?99oC" #tandard solutions $r prpard !rom commrcial solutions'

iii)

and thn introducd into clan sor%nt cartridgs" Th standard solutions $r thn analysd using th GC-,# to o%tain cali%ration cur/s' %y using introducing thm to th thrmal dsorption instrumnt (th sampl port) $hich

i/)

quippd $ith an auto-samplr" Thn' th analysis o! th EOCs !ollo$d" Th EOCs sampls $r trans!rrd !rom th cartridgs to th thrmal dsorption

/) /i)

instrumnt" Thn th systm $as run !or analysis" Th chromatographic pas illustrations $r printd out" Idnti!ication o! chromatographic pas o! th compounds $as  %asd on th rtntion tim and r!rncs to th litratur o!  mass spctrum

"+"?

Rsults 8igur  and 8igur 4 sho$s th rsults !rom th analysis o!  EOC contnt in th 3hald %rath o! to%acco cigartt and cigartt smors" Ths data could ld to a clu o! $hat compounds rtain in th rspiratory systm !rom smoing $hthr to%acco cigartt or -cigartt" Th am%int air o! th room $as compard %t$n th n/ironmnt %!or smoing and a!tr smoing in 8igur " :ccording to th discussion o! th rsults o! this 3primnt' th 3hald %rath n/ironmnt $as rich in acton and isoprn" Ths ar th typical compounds that can % idnti!id in this ind o! sampl" :s !or 8igur 4' th chromatograms o! th 3hald %rath o! th thr sampls $r compard to th sampl o! th n/ironmntal air o!  th room a!tr smoing" Th chromatogram o! th 3hald air !rom to%acco cigartt sho$d a mor simpli!id mi3tur o! compounds'  %sids lo$ring in th a%undanc o! som compound as compard to th smo chromatogram" This indicats that most o! th compounds o! th smo $r rtaind in th rspiratory systm" &hil !or th cigartt 3hald %rath chromatograms' %oth Typ  and Typ 4 sho$d th a%snc o! propyln glycol and glycrin pas' $hich indicats that ths compounds $r rtaind in th lungs" sids that' th chromatogram o! th to%acco cigartt 3hald %rath sho$d a signi!icant di!!rnc to th chromatograms o!

th

-cigartt 3hald

%rath"

On

th

othr

hand'

th

chromatograms o! %oth Typ  and Typ 4 -cigartt 3hald %rath $r almost similar" Comparing to%acco cigartt and -cigartt 3hald %raths' to%acco cigartt chromatograms illustratd a highr   %urdn o! EOCs than -cigartt"

LIMITATIONS

HPLC INTRODUCTION 5igh-pr!ormanc liquid chromatography' 5ALC is a trm that can % usd intrchanga%ly $ith LC' liquid chromatography" Th trm $as usd to distinguish th mthod it rprsnts $ith th classic gra/ity-!lo$ liquid chromatography mthod" Th gnral instrumntation componnts consistd in LC is %asically similar to th GC' %ut thy di!!r in th typs o! th componnts usd' $hich $ill latr % 3plaind" Liquid chromatography historically %gan $ith th us o! glass column $ith diamtrs o! 9  1 9mm" Th stationary phas consistd o! solid particls' pacd $ith 9 1 99cm lngth" Th particls $r coatd $ith an adsor%d liquid" During that tim' s/ral modi!ications to th si. o! th particls $r mad to sta%lish a rasona%l !lo$ rats ho$/r !aild'  %caus thy ld to longr tim consumption" Thn' attmpts to spd up th partition procss $as also mad %y putting on prssurs' %ut ld th incras o! th plat hight' $hich $as in!!icint" Liquid chromatography startd to d/lop $hn scintists rali.d that th pr!ormanc o!  th column can % %oostd %y dcrasing th si. o! th particls o! pacings" Thn' in lat *9s' th tchnology to produc pacings $ith particls o! diamtr o! ? 1 9>m $as d/lopd" Th instrumntation o! LC %cam mor sophisticatd in contrast to th traditional gra/ity-!lo$ liquid chromatography' as th d/lopmnt o! th pacing particls rquird th opration o! th systm to % pr!ormd at high prssurs" LC is popularly usd in analytical sparation tchniqus du to its ad/antags" Ths ad/antags includ0 highr snsiti/ity' adapta%l to accurat quantitati/ dtrminations' asy to handl and suita%l !or sparation o! non-/olatil spcis" On top o! thos' th $idsprad o! th instrumnt is applica%l to su%stancs that ar important in th industry' many !ilds o!  scinc and th pu%lic" Th su%stancs includ0 hydrocar%on' drugs' protins' car%ohydrats'  psticids' stroids' anti%itics and /arious inorganic su%stancs"

T56OR7 Th typical componnts o! instrumnts in LC ar sho$n in 8igur " In modrn LC' prssurs ar pumpd at s/ral hundrd atmosphrs" This is rquird in ordr to achi/ rasona%l !lo$ rats !or pacing o! ? 1 9>m" 5nc' th 5ALC quipmnt is mor compl3 and costly compard to othr typs o! chromatographic instrumnts" Th instrumntations o! a gnral 5ALC ar la%oratd %lo$" ,o%il phas rsr/oirs and sol/nt tratmnt systm@ In a modrn LC apparatus' it may consist o! singl or multipl glass rsr/oirs" Th rsr/oirs ar containd $ith 99mL or mor liquid sol/nts" Arsnc o! dissol/d gass and dust !rom liquid sol/nts could intr!r th !lo$ rats and %and sprading' as $ll as th dtctor   pr!ormanc" Thus' som rsr/oirs ha/ a %uilt in sparging systm' as sho$n in th !igur" Th spargr !unctions as dissol/d gasss rmo/r %y th mans o! !in %u%%ls o! inrt gas that is insolu%l in mo%il phas" Othr systms that may % %uilt in th dgassr ar /acuum  pumping systm' a distillation systm' a d/ic !or hating and stirring' or as sho$n in th !igur' th inlt !iltr" It !unctions as a !iltration to th dust and particulat mattr in th sol/nts $hich can caus damag to th pumping systm and in2ction systm' and could also clog th column" In addition' partitioning /al/s ar also quippd into a modrn LC instrumnts to hlp th partitioning o! th sol/nt introducd in 5ALC at ratios that can % /arid continuously" Aumping systm@ Aumps ar rquird in LC to0 i) ii) iii) i/) /)

Gnrat prssurs to up to 999psi Aroduc puls-!r output Rgulat !lo$ rats at th rang o! 9" to "9 mL;min : %ttr !lo$ rproduci%ility Control corrosion %y sol/nts"

Though pump usd to incras th prssurs in LC' %ut ths high prssur liquids do not caus any ha.ardous 3plosion' sinc liquids ar not /ry comprssi%l" Th rsults o! ths high prssurs may only lad to componnt ruptur' and consquntly caus sol/nt laag" Instad' this laag may constitut to a !ir and may % ha.ardous to th n/ironmnt"

Th $idly usd pumps typs in LC ar th scr$-dri/n syring and th rciprocating pump" In most commrcial LC instrumnts' thy ar quippd $ith a computri.d systm in $hich thy ar mor con/nint in controlling th masurmnt o! th !lo$ rat" #ampl-in2ction systm #ampl introducd into th column pacing is rquird to % at a /ry small /olum" Th /olum rang may % o! a !$ tnths o! microlitr to o! a%out 99>L" This hlps to rduc th limiting !actor in LC prcision' $hich is th rproduci%ility $ith $hich sampl can % introducd into th column" Th common consr/ati/ $ay to introduc th sampl into LC is %y using sampling loops" Thy ar intgratd as a part o! th LC instrumnt and th loops can also % intrchanga%l dpnding on th si.s o! th sampl" Ths loops pro/id choics !or sampl si.s ranging !rom >L to 99>L" On th othr hand' most LC instrumnts in this modrn day ar quippd $ith auto-in2ctors systm" Th systm has sampling loops and syring pump $hich allo$ in2ction /olums to % ranging !rom lss than >L to mor than mL" &hil som systms allo$ !or controlling o! th n/ironmnt tmpratur o! th sampling port and som may %  programma%l to allo$ !or unattndd in2ctions" Columns@ Thr ar s/ral typs o! LC columns in/ntd" Ths columns di!!r in trms o! physical constructions' si.s' pacing and costs" Usually' thy ar %uilt !rom smooth-%or stainlss stl tu%ing' somtims ha/y-$alld glass tu%ing and polymr tu%ing' and thy /n %uilt !rom stainlss stl' lind $ith glass or polymr" Common typs o! columns ar0 analytical columns and guard columns" Column tmpratur control@ #om applications o! LC do not rquir a clos control o! tmpratur" Thy opratd at room tmpratur" ut tmpratur rgulation is o!tn ncssary to o%tain %ttr chromatograms" ,ost ad/ancd instrumnts no$ ar quippd $ith hatrs to rgulat th tmpratur o! th column" sids that' som columns may also % !ittd $ith $atr 2act' $hich !unctioning as th tmpratur rgulator" Column pacing@

In 5ALC' thr ar t$o $ll-no$n typs o! column pacing usd0 Allicular particl 1 sphrical' nonporous' glass or polymr %ads $ith typical diamtrs o! ?9  1 +9>m" Thr $as a thin' porous layr dpositd on th sur!ac o! th %ads" This layr may  % mad !rom silica' alumina' or an ion-3chang rsin" Aorous particl 1 consists o! porous microparticls $ith diamtrs ranging %t$n ? 1 9>m" Th particls ar mad !rom alumina' ion-3chang rsin' or th most commonly usd0 silica" Th silica particl o!tn coatd $ith thin organic !ilms" Th !ilms ar chmically or  physically %ondd to th sur!ac" Dtctors Th idal proprtis o! dtctor usd in LC includ0 i) ii) iii) i/) /) /i)

:dquat snsiti/ity Good sta%ility and rproduci%ility Linar rspons to soluts that 3tnds o/r s/ral ordrs o! magnitud #hort rspons tim indpndnt o! !lo$ rat ,inimal intrnal /olum to rduc .on %roadning Compati%l $ith liquid !lo$"

Th dtctors commonly usd in 5ALC ar0 UE-Eisi%l a%sorption dtctors' !luorscnc dtctors' r!racti/-ind3 dtctors' and mass spctromtric dtctors" In gnral' th analysis run through th 5ALC is quit similar to th analysis %y GC systm" Th sampl is !irstly sampl port o! th systm' th mo%il phas $ill carry th analyts o!  th sampl to th column o! th 5ALC systm !or sparation' thn th output !rom th column is !d into th dtctor d/ic" 8inally' a schmatic illustration' $hich is th outcom o! th analysis' is printd out o! th output systm"

#:,AL6 AR6A:R:TION : rsarch o! idnti!ication and quanti!ication o! %ioacti/ compounds in co!! %r$s $as conductd %y Naira Aornr Rodrigus and Nura ragagnolo" 5igh Ar!ormanc Liquid Chromatography (5ALC) $as in/ol/d in th mthodology o! th analysis" Thr $r s/ral typs o! sampls to % idnti!id and quanti!id in th analysis" Th sampls $r acquird !rom th local mart in th city o! Campina' #an Aaulo' ra.il" Th sampls $r also producd o! di!!rnt manu!acturrs" :s dscri%d in thir pacaging' th sampls consistd o! di!!rnt dgr o! idntity and quality' $hich mad thm to % classi!id into gourmt' traditional and suprm" sids that' thir dgr o! roasting also rangd %t$n light to dar" Th typs o! th sampls includ0 J rgular roastd ground co!!s (mard as RR)' ? dca!!inatd roastd ground co!!s (DR)' 4 rgular solu%l co!!s (R#)' and 4 dca!!inatd solu%l co!!s (D#)" Th roastd ground co!!s and th solu%l co!!s $r %r$d and prpard %y th ratio o! co!! to $atr as rcommndd in th pacaging" #ampl Arparation Tchniqus@ i) ii)

iii) i/) /)

To prpar th roastd ground co!!s %r$' g o! th sampls $r !irstly $ightd in a !iltr papr &hatman no" + 9 mL o! ultrapur $atr o! a%out *4 oC $as slo$ly pourd through th cntr o!  th !iltrpapr" :nd thn' th solution $as trans!rrd into /olumtric !lass" Th !inal /olum o! th %r$ o! roastd ground co!!s /arid !rom ?+-?JmL" To prpar th solu%l co!!s %r$' 9"4g o! th sampls $r $ighd' and trans!rrd into 9mL /olumtric !lass" Thy $r dissol/d %y ultrapur $atr at tmpratur o! 4oC up to th cali%ration mars o! th !lass" Th %r$s o! th sampls $r immdiatly !ro.n %y liquid nitrogn" :nd thn' thy $r stord in a !r.r at th tmpratur o! -H9oC until analysis"

6:,AL6 O8 :N:L7#I# :nalysis o! %ioacti/ compounds in co!! %r$s %y 5ALC Th sampl prparation mthod $as listd in th sampl prparation part ' sction %rapa  point %rapa" ,6T5ODOLOG7 Th analysis procdurs $r quit compl3' thus' this part is only !ocusing on th mthodology on using th 5ALC" Lin to th !ull /rsion o! th articl can % !ollo$d in th r!rncs" Componnts to % analysd@ ca!!in' chlorognic acids (CG:) and dri/ati/s' trigonllin' nicotinic acids' -hydro3ymthyl!ur!ural' tho%romin' thophyllin" #tandard solutions prpard !or sampl idnti!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)' trigonllin hydrochlorid (*HB purity)' nicotinic acid (**"B  purity)' -hydro3ymthyl!ur!ural (**B purity)' tho%romin (**B purity)' thophyllin (**B  purity)" Th opration o! th 5ALC systm0 ,odl@ #himad.u 5ALC' quippd $ith %inary pump' dgassr $ith hlium' and automatic in2ction systm" 8or quantitati/ analysis' th 5ALC systm $as coupld to a diod array dtctor (D:D)' $hil !or compound idnti!ication purpos' it $as coupld $ith th mass spctromtr (,#)" ,o%il phas@ mi3tur o! H9B (/;/) 9m, citric acid (p5 4)' 49B (/;/) mthanol and 99B (/;/) mthanol !or ca!!in and CG: and its dri/ati/s analysis" &hil !or th othr  compounds analysis' th mo%il phas includs0 9"B (/;/) actic acid (p5 ?) and mthanol" Columns@ 8or ca!!in and CG: and its dri/ati/ analysis' OD#-CH #him-pac column o!  m' 4+9mm  +"mm innr diamtr $as usd" &hil th othr analysis' th sam typ o!  column $as also usd" 63primntal Arocdurs@

i)

Th co!! %r$s $r tratd !irstly to prcipitat th compounds o! highr  molcular

ii)

$ight

%y

using

.inc

actat

solution

and

potassium

h3acyano!rrat solution" 8or ca!!in' and CG: and its dri/ati/s dtrmination'  mL o! ach co!!  %r$s $r trans!rrd into tst tu%s' and thn addd $ith 4mL o! ultrapur $atr" N3t' 9"mL o! .inc actat solution and potassium h3acyano!rrat

iii)

solution' and 9"HmL o! mthanol $r addd into th %r$ solution" Th mi3turs $r shan !or ?9 sconds' and thn l!t to sttl !or 9

i/)

minuts" Th prcipitat $as sparatd !rom th mi3turs %y cntri!ugation" Th

/)

suprnatants $r collctd' and thn !iltrd using 9"+>m mm%ran !iltrs" Th analysis $as carrid out using 5ALC-D:D and 5ALC-D:D-,#" !or analysing th sampls' standard solutions $r analysd in th systms %y introducing thm to th auto-samplr o! th systm" Thn' analysis o! th

/i)

sampls !ollo$d" &hil !or th othr compounds dtrmination' th %r$s $r tratd using

/ii) /iii)

9"4mL o! aquous lad actat solution" Th analysis using 5ALC systms !ollo$d" Th mass spctra illustrations $r printd out and discussd quantitati/' and qualitati/ly %y comparing th spctra to th litratur"

R6#ULT# Idnti!ication o! ca!!in and CG: and dri/ati/s@

Idnti!ication o! trigonllin' nicotinic acids' -5,8' tho%romin and thophyllin"

8igur  sho$s th chromatogram !or th analysis o! ca!!in and CG: and dri/ati/s" asd on $hat had %n rportd in th analysis' %sids ca!!in' thr $r 4 dri/ati/s o! hydro3ycinnamic" Thy $r classi!id into + groups0 CG: (pas ' 4' +' ' J' ' 4' H' *' 49' 4' 44' 4?' 4' 4)' chlorognic acid lactons (pas 9' ?' ' J)' cinnamoyl-amino acid con2ugats (pas 4+' 4J) and !r cinnamic acid (pa H)" y comparison to th UE-/isi%l and mass spctra o! authntic standards' th compounds o!  trigonllin' nicotinic acids' -5,8' tho%romin and thophyllin $r idnti!id in th chromatogram in 8igur 4" Th quanti!ication $as also don to !ind th amount o! %ioacti/ compounds contnt in th co!! %r$s' and th rsults $r summari.d as in Ta%l "

LIMITATIONSREFERENCES ''**+,%./-,%,3*S00214"!1501021*1-/2%0-S00214"!15010213+6%789'6:100!1-75-115-4+100000++;0702&+,+':1##00#4#091!;5!4!1#040257#+41!#50;# .,2- ''**+,%./,%,3%%?6'3%?%3>*S0415!510011""*1-/2%0S0415!510011""-3+6%789'6:++2;"-4027-115-+;700000++,;"1&+,+':1##04!149,2@+!,20;4"##07++!,+;""2!