Phoebe Jilliane O.

Lacson FS 135
Anna Margarita P. Tongco Written Report
I. Introduction
A. Brief History
The first no!n "se of li#"i$ chro%atograph& !as in the late 1'()s. *t is tra$itionall&
attrib"te$ to R"ssian botanist Mihail Ts!ett+ !ho "se$ it to separate an$ isolate plant
pig%ents ,-etanco"rt . /ottlieb+ 0)1)1. *n his research of chloroph&ll+ Ts!ett "se$ col"%ns
of calci"% carbonate to separate plant co%po"n$s. The pig%ents !ere $issol2e$ in a li#"i$
an$ po"re$ onto the col"%n+ !hich !as contin"all& !ashe$ !ith the li#"i$. 3e then
obser2e$ the separation of the pig%ents into colore$ ban$s as the& tra2elle$ $o!n the
col"%n. -eca"se of the color ban$s pro$"ce$ on the absorbent be$+ he "se$ the ter%
chro%atograph& 4 5color !riting6 ,Fit7geral$+ 1((81.

Fig"re 1. Ts!ett9s chro%atograph& set:"p.
Ts!ett presente$ the %etho$ in 1()3+ at the 11
;ongress of <at"ralists an$ =octors+
hel$ in St. Petersb"rg. =espite the significance of his !or+ beca"se it !as presente$ in
R"ssian+ his $e2elope$ techni#"e !as not rea$il& accepte$ ,/ilbraith Laboratories+
0)111.Wi$esprea$ "se began onl& in the earl& 1(3)s+ !hen other scientists $escribe$ their
!or on plant pig%ents+ incl"$ing carotenoi$s an$ >antoph&lls ,-raith!aite . S%ith+ 1(((1.
There !as %ini%al $e2elop%ent in the fiel$ of li#"i$ chro%atograph& in the 1(?)s@
instea$+ other relate$ fiel$s s"ch as col"%n chro%atograph&+ gas a$sorption chro%atograph&+
an$ gas:li#"i$ partition chro%atograph& $e2elope$ fro% the fo"n$ation lai$ b& li#"i$
One %ore i%portant e2ent in the histor& of li#"i$ chro%atograph& !as the a!ar$ing of
the 1(50 <obel Pri7e for ;he%istr& to -ritish bioche%ists Archer Martin an$ Richar$ S&nge
,-etanco"rt . /ottlieb+ 0)1)1. Martin an$ S&nge $e2elope$ the %etho$ !herein the
stationar& phase 4 !ater 4 !as s"pporte$ on silica gel an$ "se$ to separate ac&late$ a%ino
aci$s ro% protein h&$rol&sates. Fro% this e>peri%ent+ the& establishe$ the principles of
partition chro%atograph&. The& also intro$"ce$ the concept of high:perfor%ance li#"i$
chro%atograph&+ s"ggesting that the "se of s%all particles an$ high press"re in L; testing
co"l$ i%pro2e separation ,/albraith Laboratories+ 0)111.
Mo$ern $e2elop%ents in engineering an$ %icroelectronics o2er the last $eca$es ha2e
enable$ %an"fact"res to pro$"ce co%ple> a"to%ate$ instr"%ents that achie2e repro$"cible
chro%atograph&. These %achines are able to not onl& set:"p an$ control the instr"%ents+ b"t
collect an$ process the $ata to pro$"ce chro%atogra%s an$ anal&tical reports ,-raith!aite .
S%ith+ 1(((1.
B. Principes
The *APA;9s 1((3 $efinition of chro%atograph& $efines it as 5a ph&sical %etho$ of
separation in !hich the co%ponents to be separate$ are bet!een t!o phases+ one of !hich is
stationar& !hile the other %o2es in a $efinite $irection.6 As s"ch+ chro%atograph&
enco%passes a n"%ber of 2ariations+ on the basic principle of separation of co%ponents in a
%i>t"re achie2e$ b& a s"ccessi2e series of e#"ilibri"% stages. These e#"ilibria $epen$ on the
$ifferential $istrib"tion of in$i2i$"al co%ponents bet!een the t!o phasesB %obile an$
stationar& ,-raith!aite . S%ith+ 1(((1. The co%ponents !ithin the %i>t"re to be anal&se$
are separate$ base$ on each co%ponent9s affinit&. Affinit& is establishe$ $epen$ing on
$ifferences in a$sorption+ ion:e>change+ partitioning+ or si7e. These $ifferences allo! the
%i>t"re co%ponents to be separate$ fro% each other+ an$ !ill also $eter%ine the transit ti%e
of these sol"tes ,Fit7geral$+ 1((81.
="e to these $ifferences+ the co%po"n$s are separate$ into $istinct ban$s !ithin the
col"%n. *n a$$ition+ if the co%ponents are of $ifferent polarities an$ the %obile phase has a
$istinct polarit&+ certain co%ponents !ill %igrate faster than others ,-etanco"rt . /ottleib+
0)1)1. The basis of #"antitation is the e#"ilibration of the each anal&te bet!een the %obile
an$ stationar& phase ,Cebbe"s+ 0))D1. Once the sa%ple has e#"ilibrate$ 4 that is+ it no
longer tra2els $o!n the col"%n bet!een %obile an$ stationar& phase 4 it can be #"antitate$
an$ i$entifie$ "sing the color of the for%e$ ban$s an$ the $istance !hich the& tra2el.
Fig"re 0. Sche%atic $iagra% e>plaining %o2e%ent of co%ponents in a chro%atographic
Th"s+ li#"i$ chro%atograph& refers to an& chro%atographic proce$"re in !hich the
%o2ing phase is a li#"i$. The nat"re of the correspon$ing stationar& phases allo! for
$ifferentiation into t!o classifications of li#"i$ chro%atograph& ,Cat7 et al.+ 1(('1. The
stationar& phase can be li#"i$ or soli$. When a sol"te is partitione$ bet!een a li#"i$ %obile
phase an$ a li#"i$ fil% stationar& phase+ the %etho$ is no!n as partition chro%atograph&+
or li#"i$:li#"i$ chro%atograph& ,LL;1. When a ph&sical s"rface forces control on the
retention properties of the co%ponent on a soli$ stationar& phase+ the %etho$ is no!n as
adsorption chro%atograph&+ or li#"i$:soli$ chro%atograph& ,LS;1 ,-raith!aite . S%ith+
Fig"re 3. =ifferent t&pes of li#"i$ chro%atograph& $epen$ent on sa%ple affinit&.
The li#"i$ chro%atograph& techni#"es !hich !ere applie$ in foo$ anal&sis !ere paper
chro%atograph&+ thin la&er chro%atograph& ,TL;1 ,both of these techni#"es %a& be referre$
to as planar chro%atograph&1+ an$ col"%n li#"i$ chro%atograph& ,<ielsen+ 0)1)1.
II. Met!ods of Li"uid C!ro#$to%r$p!y
A. P$per C!ro#$to%r$p!y
1.1 Principle
*n paper chro%atograph&+ the stationar& phase is a li#"i$ ,!ater1 an$ the %obile phase
is also a li#"i$ ,organic sol2ent E !ater1. For this reason+ paper chro%atograph& is also calle$
li#"i$:li#"i$ chro%atograph& or partition chro%atograph& ,=a2i$+ 0))1@ <ielsen+ 0)1)1.
Anal&tes are separate$ base$ on the interactions bet!een t!o non:%iscible li#"i$ phases
accor$ing to their relati2e sol"bilit& ,Raa%an+ 0))81. Paper generall& ser2es as a s"pport for
the li#"i$ stationar& phase. The $issol2e$ sa%ple is applie$ as a s%all spot or strea one half
inch or %ore fro% the e$ge of a strip or s#"are of filter paper ,"s"all& cell"lose1+ !hich is
then allo!e$ to $r&. The $r& strip is s"spen$e$ in a close$ container in !hich the at%osphere
is sat"rate$ !ith the $e2eloping sol2ent ,%obile phase1+ an$ the paper chro%atogra% is
$e2elope$ ,<ielsen+ 0)1)1. The en$ closer to the sa%ple is place$ in contact !ith the sol2ent+
!hich then tra2els "p or $o!n the paper b& capillar& action ,$epen$ing on !hether ascen$ing
or $escen$ing $e2elop%ent is "se$1+ separating the sa%ple co%ponents in the process. When
the sol2ent front has tra2ele$ the length of the paper+ the strip is re%o2e$ fro% the $e2eloping
cha%ber an$ the separate$ 7ones are $etecte$ b& an appropriate %etho$. The stationar& phase
in paper partition chro%atograph& is "s"all& !ater. The s"pport %a& be i%pregnate$ !ith a
nonpolar organic sol2ent an$ $e2elope$ !ith !ater or other polar sol2ents or !ater ,re2erse$:
phase paper chro%atograph&1 ,<ielsen+ 0)1)1. 3o!e2er+ anal&tes are separate$ base$ on the
interactions bet!een t!o non:%iscible li#"i$ phases accor$ing to their relati2e sol"bilit&
,Raa%an+ 0))81. Paper chro%atogra%s are $e2elope$ to a pre$eter%ine$ $istance that allo!s
a calc"lation of the R
2al"e of each co%ponent after $e2elop%ent ,Ah"Fa+ 0))31. The res"lts
of paper chro%atograph& are e>presse$ in ter%s of R
2al"es+ i.e.+ retar$ation factors ,Fig.31.
Fig"re ?. R
2al"es in paper chro%atograph&

The separate$ co%ponents are i$entifie$ generall& on the basis of their %obilit& in the
%obile phase. This %obilit& is e>presse$ b& R
2al"e ,Ah"Fa+ 0))31. The $istance %o2e$ b&
the sol2ent fronts+ A an$ - ,$
+ $
+ $
1 are %eas"re$ in centi%eters+ fro% the line !here the
sa%ple %i>t"re !as spotte$ for chro%atograph&. For sa%ples A an$ -+ the center of the spot
is to be consi$ere$ for %eas"ring the $istances the& tra2elle$. <ote that the co%po"n$s A an$
- are retar$e$ !ith respect to the sol2ent front ,Fig. 31. The R
2al"e of a co%po"n$ is a
ph&sical constant an$ $epen$ent on the nat"re of the stationar& phase ,paper gra$e+ thicness+
$ensit&1 an$ also on the nat"re of the %obile phase. 3ence+ !here2er R
2al"es are gi2en+ one
%"st also %ention the nat"re of the stationar& phase an$ %obile phase. Paper chro%atograph&
techni#"es !ere classifie$ intoB ,11 Ascen$ing Paper ;hro%atograph&+ ,01 =escen$ing Paper
;hro%atograph& an$ ,31 3ori7ontal Paper ;hro%atograph& ,=a2i$+ 0))11.
1.0 *nstr"%entation
The instr"%ent or apparat"s re#"ire$ for paper chro%atograph& consists of an air tight
cha%ber+ a sol2ent tro"gh an$ a s"pport of paper ,Raa%an+ 0))81.
*n ascen$ing techni#"e+ the sa%ple is spotte$ G in fro% the en$ of a paper strip. The
paper is s"spen$e$ 2erticall&+ in a chro%atographic cha%ber+ !ith the spotte$ en$ $o!n+ an$
allo!e$ to $ip into abo"t H in of a sol2ent. The sol2ent rises "p the paper b& capillar& action+
effecting a separation of the co%ponents as it ascen$s ,Fig. ?1 ,Po%eran7 . Meloan+ 0)))1.
3o!e2er+ the sol2ent !ill rise onl& "p to 0) to 05 c% an$ separations of the anal&tes are
li%ite$. The resol"tion of sa%ple b& ascen$ing techni#"e is better co%pare$ to the
$escen$ing techni#"e. This $"e to the forces !hich are acting on the sol"te in the ascen$ing
techni#"e ,Raa%an+ 0))81.

Fig"re 5. Apparat"s for ascen$ing paper chro%atograph& ,Po%eran7 . Meloan+ 0)))1
*n contrast+ $escen$ing techni#"e in2ol2es the spotting of the sa%ple 0:3 in fro% the
en$ of a paper strip. The strip is s"spen$e$ 2erticall& in a chro%atographic cha%ber !ith the
spotte$ en$ "p. The 0: to 3:in !ic is then bent o2er an$ place$ in a sol2ent tan. After the
sol2ent rises "p the !ic+ it $escen$s across the spot an$ $o!n the strip. An antisiphon bar is
place$ at the top of the strip to pre2ent the sol2ent fro% si%pl& siphoning o"t of the tan ,Fig.
51. This techni#"e per%its a longer strip to be "se$ than !ith ascen$ing chro%atograph&+ an$
allo!s a correspon$ing increase in resol"tion ,Po%eran7 . Meloan+ 0)))1.

Fig"re 8. Apparat"s for $escen$ing paper chro%atograph& ,Po%eran7 . Meloan+ 0)))1
*n hori7ontal techni#"e+ the sa%ple is place$ at the center of a circ"lar:shape$ paper.
The %obile phase sol2ent is place$ at the botto% of a circ"lar cha%ber. The spotte$ paper is
hel$ hori7ontall& on the cha%ber. The sol2ent for $e2elop%ent is applie$ at the spot b& a
capillar& t"be or !ic+ ,!hich is c"t portion of the circ"lar paper1 fro% !hich it sprea$s o"t
ra$iall&. The co%po"n$s in the %i>t"re sprea$ into a series of concentric ban$s ,Fig. 81. *n
the $escen$ing an$ ascen$ing paper chro%atograph& %etho$s+ the co%po"n$s in the %i>t"re
appear as circ"lar or o2al spots after separation ,=a2i$+ 0))11.
Fig"re D. Apparat"s an$ ;hro%atogra% for hori7ontal paper chro%atograph& ,=a2i$+ 0))11
1.3 Applications
*n foo$ in$"str&+ paper chro%atograph& is "s"all& "se$ for the separation of highl&
polar co%po"n$s s"ch as s"gars+ a%ino aci$s+ an$ nat"ral pig%ents ,Raa%an+ 0))81.
1.? A$2antages an$ Li%itations
;o%pare$ to col"%n li#"i$ chro%atograph&+ the paper chro%atograph& offers
si%plicit&+ lo! cost+ an$ "natten$e$+ hassle:free operation. *t can be r"n in 2ario"s %o$es+
an$ #"antitation %a& be achie2e$ !itho"t the "se of e>pensi2e instr"%entation ,Ah"Fa+ 0))31.
3o!e2er+ the %ain a$2antage of the ascen$ing techni#"e is the 2er& si%ple e#"ip%ent
re#"ire%ent. On the other han$+ its $isa$2antage is that the sol2ent !ill rise onl& b& ':( in an$
it is 2er& slo!+ !hereas the $escen$ing techni#"es are %"ch faster ,Po%eran7 . Meloan+
0)))@ Raa%an 0))81.
B. T!in L$yer C!ro#$to%r$p!y &TLC'
0.1 Principle
A thin la&er of the stationar& phase is for%e$ on a s"itable flat ,"s"all& a glass plate1
coate$ !ith a thin fil% of the a$sorbent ,Wilson . Waler+ 0)))@ A7ares et al.+ 0))51.
A$sorbents incl"$e silica gel+ al"%ina an$ cell"lose po!$er ,A7ares et al.+ 0))51. A sol"tion
of the %i>t"re is spotte$ on the plate thinl& an$ the %o2e%ent of the %obile phase across the
la&er is carrie$ o"t in an ascen$ing or$er b& capillar& action ,Wilson . Waler+ 0)))@ A7ares
et al.+ 0))51. As the %obile phase %o2es across the la&er fro% one e$ge to the opposite+ it
transfers an& anal&tes place$ on the la&er at a rate $eter%ine$ b& their $istrib"tion
coefficients+ C
+ bet!een the stationar& an$ %obile phases. Anal&te %o2e%ent ceases either
!hen the %obile phase ,sol2ent front1 reaches the en$ of the la&er an$ capillar& action flo!
ceases or !hen the plate is re%o2e$ fro% the %obile phase reser2oir. The %o2e%ent of the
anal&te is e>presse$ b& its retar$ation factor ,Wilson . Waler+ 0)))1B

The efficienc& of a thin la&er plate is e>presse$ b& its n"%ber of theoretical plates+ <. an$
plate height+ 3+ !here d
is the $istance %o2e$ b& the anal&te fro% the origin an$ w is the
!i$th of the spot an$ the capacit& factor+ k’+ for the anal&te+ is gi2en !here d
is the $istance
%o2e$ b& the sol2ent front fro% the origin ,Wilson . Waler+ 0)))1.

3o!e2er+ $ifferent co%ponents !ill ha2e $ifferent R
2al"es $epen$ing on their
properties. The spots are 2is"ali7e$ b& ph&sical ,nae$ e&e+ AI ra$iation1 or che%ical ,*0
2apo"r+ spra& reagents1 %eans. A special t&pe of TL; is the preparati2e TL; in !hich the
plates "se$ are bigger an$ the absorbent applie$ is thicer ,A7ares et al.+ 0))51.
A glass plate is sprea$ !ith a thin la&er of silica gel. A sa%ple is a$$e$ at one en$. A
b"ffer creeps fro% the sa%ple si$e to the opposite en$ carr&ing sol"ble %olec"les along.
Molec"les are $eposite$ base$ on the si7e ,Fig. D1 ,Raa%an+ 0))81.

Fig"re '. Apparat"s of Thin La&er ;hro%atograph& ,Raa%an+ 0))81
0.3 Applications
TL; is applie$ in %an& fiel$s+ incl"$ing en2iron%ental+ clinical+ forensic+
phar%ace"tical+ foo$+ fla2ors+ an$ cos%etics. Within the foo$ in$"str&+ TL; %a& be "se$ for
#"alit& control. For e>a%ple+ corn an$ pean"ts are teste$ for aflato>insJ%&coto>ins prior to
their processing into corn %eal an$ pean"t b"tter+ respecti2el&. Applications of TL; co%prise
the anal&sis of a 2ariet& of co%po"n$s+ incl"$ing lipi$s+ carboh&$rates+ 2ita%ins+ a%ino aci$s+
an$ nat"ral pig%ents ,<ielsen+ 0)1)1.
0.? A$2antages an$ Li%itations
The a$2antages of thin la&er chro%atograph& areB ,11 the separation $epen$s "pon the
%at"re of the chro%atographic %e$ia "se$+ it can be achie2e$ either b& partitioning+
a$sorption+ ion e>change or %olec"lar sie2ing pheno%enon !hile in paper chro%atograph&+
the separation is base$ on partitioning onl&+ ,01 The %etho$ is e>tre%el& rapi$ as the
separation can be achie2e$ !ithin an ho"r or so !hile in paper chro%atograph& it %a& tae "p
to abo"t ':0? ho"rs+ ,31 *t is relati2el& %ore sensiti2e since lo!er concentrations of
co%po"n$s in the %i>t"re can be s"ccessf"ll& separate$ an$ $etecte$ an$ ,?1 corrosi2e agents
lie 3
an$ high te%perat"res can be "se$ to locate the separate$ co%po"n$s !hich is not
possible in paper chro%atograph& ,S!a%&+ 0))'1.
A li%itation of TL; is that once the sa%ple has been loa$e$ onto the plate+ it cannot
be reco2ere$. This %"st be taen into acco"nt if onl& s%all a%o"nts of sa%ple are a2ailable
for testing. Another li%itation is that if TL; is to be "se$ for i$entification+ certain R 2al"es
,base$ on %igration of spots1 %"st be no!n beforehan$ ,Ani2ersit& of Toronto+ 0)1)1.
C. Cou#n Li"uid C!ro#$to%r$p!y
3.1 Principle
*n col"%n li#"i$ chro%atograph&+ the %obile phase is li#"i$ an$ the stationar& phase
can be either soli$ or li#"i$ s"pporte$ b& an inert soli$. ,<ielsen+ 0)1)1. The sa%ple %i>t"re
is passe$ thro"gh a col"%n pace$ !ith soli$ particles+ !hich %a& or %a& not be coate$ !ith
a li#"i$. =epen$ing on the sol2ents an$ pacing con$itions+ the co%ponents in the sa%ple
!ill tra2el the col"%n at $ifferent spee$s+ res"lting in separation ,Sn&$er . Cirlan$+ 1(D31.
There are certain factors !hich %"st be consi$ere$ in col"%n li#"i$ chro%atograph&.
First+ the col"%n $i%ensions $epen$ on the #"alit& of %aterial to be separate$. Also+ in or$er
to obtain %a>i%"% efficienc&+ the col"%n %"st be e2enl& pace$+ to %ini%i7e $istortion of
chro%atographic bo"n$aries an$ pre2ent channeling. Another factor to be consi$ere$ is the
particle si7e of the pacing %aterial. *f the particle si7e is "nifor%+ ho%ogeneo"s pacing is
%a$e %ore possible ,-raith!aite . S%ith+ 1(((1. 3o!e2er+ it %"st be note$ that changing
particle si7e can also affect the press"re !ithin the col"%n+ so this %"st be controlle$ as !ell.
Te%perat"re also affects the li#"i$ chro%atographic separationsB at higher te%perat"res+
sol2ent 2iscosit& $ecreases+ allo!ing for easier flo! of el"ent. The rate of sol"te $iff"sion
also increases+ ca"sing faster $iff"sion ,R"binson . R"binson+ 0)))1.
A ( B ( C
Soli$ Particles
,pacing %aterial:
stat ionar& phase1
Kl"ant ,el"ate1
Sol2ent,%obile or
%o2ing phase1

Fig"re (. =iagra% of si%ple li#"i$ col"%n chro%atograph&.
3.0 T&pes of ;ol"%n ;hro%atograph& ,A7ares et al.+ 0))51B
3.0.1 Flash ;hro%atograph& 4 *t in2ol2es appl&ing of press"re on top of the col"%n
!hich enables the separation to be faster.
3.0.0 L"ic ;ol"%n ;hro%atograph& 4 *t in2ol2es the "se of 2ac""% press"re at the
botto% of the col"%n. These are the lo! press"re li#"i$ chro%atograph& ,LPL;1+ %e$i"%
press"re li#"i$ chro%atograph& ,MPL;1 an$ high press"re li#"i$ chro%atograph& ,3PL;1.
These t&pes of techni#"es gi2e better separation since the& "se longer b"t thinner col"%ns.
3.0.3 /el:per%eation or the Si7e K>cl"sion ;hro%atograph& 4 *n this %etho$+ the
stationar& phase is %a$e "p of cross:line$ pol&%er. The separation $epen$s on the abilit& of
%olec"les to penetrate the sie2e:lie str"ct"re of the gel. S%all %olec"les can penetrate the
poro"s str"ct"re %ore easil& than the larger ones. /el per%eation is "se$ in the separation of
co%ponents accor$ing to si7e or %olec"lar !eights+ specificall& in the st"$& of bio%olec"les
an$ pol&%ers. *n si7e:e>cl"sion chro%atograph&+ poro"s gels are "se$ as stationar& phases+ in
!hich separation is $"e to $ifferences in the si7e of the sol"tes. Large sol"tes are "nable to
penetrate into the poro"s stationar& phase an$ so #"icl& pass thro"gh the col"%n. S%aller
sol"tes enter into the poro"s stationar& phase+ increasing the ti%e spent on the col"%n.
3.0.? *on:K>change ;hro%atograph& ,*K;1 4 This t&pe of col"%n chro%atograph&
in2ol2es the separation of %olec"les base$ on their o2erall charge at a gi2en p3 ,Raa%an+
0))81. The stationar& phase consists of a pol&%er !ith a charge$ f"nctional gro"p. *on:
e>change resins are classifie$ accor$ing to the ionic nat"re of the f"nctional gro"p an$ the
pol&%er. This t&pe of col"%n chro%atograph& is "se$ for the separation of charge$ particles
an$ is co%%onl& "se$ in the st"$& of a%ino aci$s ,A7ares et al.+ 0))51. 3o!e2er+ stationar&
phases consisting of a soli$ s"pport !ith co2alentl& attache$ anionic ,e.g.+ 4SO341 or cationic
,e.g.+ 4<,;3313E1 f"nctional gro"ps are "se$ in ion:e>change chro%atograph&. *onic sol"tes
are attracte$ to the stationar& phase b& electrostatic forces ,3ar2e&+ 0)))1. The p"rpose of the
ion:e>change resin bea$s is to separate positi2el& charge$ proteins fro% negati2el& charge$
ones ,Raa%an+ 0))81.
3.3 *nstr"%entations
;ol"%n chro%atograph& can be applie$ in t!o %etho$sB $r& an$ !et %etho$. For the
$r& %etho$+ the col"%n is fille$ !ith a $r& po!$er+ then the %obile phase is a$$e$. The
li#"i$ is fl"she$ thro"gh the col"%n+ an$ as s"ch %"st ne2er r"n $r&. For the !et %etho$+
sl"rr& is %a$e b& %i>ing the el"ent !ith the po!$er+ !hich is then po"re$ into the col"%n.
For both %etho$s+ the %o2e%ent of the %obile phase can be thro"gh either gra2it& or e>ternal
press"re ,Ani2ersit& of ;olora$o+ 0)101.
The top of the col"%n can be protecte$ !ith %aterials s"ch as san$+ glass !ool+ or
filter paper. When the sa%ple has been entirel& a$sorbe$+ the 2oi$ can be fille$ !ith sol2ent
an$ the chro%atogra% $e2elope$. The sol2ent is replenishe$ as re#"ire$ ,-raith!aite .
S%ith+ 1(((1.
Fig"re 1). Set:"p of col"%n li#"i$ chro%atograph&.
Most li#"i$ chro%atograph& set:"ps also ha2e $etectors+ !hich can better i$entif& the
separation bet!een co%ponents. Most $etectors ha2e 2ol"%es in the range of 5:0) ML 4 the
2ol"%e is ept s%all to $ecrease the effects of el"tion %i>ing. Kl"tion %i>ing occ"rs !hen
the el"ent 2ol"%e !ithin the $etector cell is %i>e$ b& the flo! of li#"i$+ inter%i>ing the
earlier an$ later 2ol"%es. Kach ban$ is th"s broa$ene$ b& this %i>ing+ an$ a larger cell !ill
ha2e a larger el"ent 2ol"%e that can be %i>e$. Th"s the $etector cell is ept s%all to pro$"ce
onl& a %inor broa$ening of ban$s ,R"binson . R"binson+ 0)))).
3.? Applications
Man& of the applications of col"%n chro%atograph& ha2e been replace$ b& 3PL; or
thin:la&er chro%atograph&. A"to%ate$ progra%s ha2e also been $e2elope$ !hich
significantl& ease an$ spee$ "p the anal&tical process of chro%atograph& ,Fair . Cor%os+
0))'1. 3o!e2er+ it still has applications in large:scale separations ,larger than 1) g1 of
reaction %i>t"res in s&nthetic organic che%istr&. /el an$ si7e:e>cl"sion chro%atograph& are
also still "se$ e>tensi2el& in bioche%istr&+ especiall& in separation of n"cleic an$ a%ino aci$s
,-raith!aite . S%ith+ 1(((1. The %ain principles of col"%n chro%atograph& are also "tili7e$
in constr"cting co%ponent profiles of 2ario"s %aterials+ incl"$ing foo$s an$ be2erages
,C"s%iere . -al$+ 0))'1.
3.5 A$2antages an$ Li%itations
;ol"%n chro%atograph& is a p"rification techni#"e+ isolating $esire$ co%po"n$s
fro% a single %i>t"re. *ts %ain a$2antage is that it can be easil& a$F"ste$ $epen$ing on !hat
co%po"n$s are being anal&se$ forB factors s"ch as te%perat"re+ sol2ent+ particle si7e+ an$
%etho$ are all rea$il& a$F"stable+ as co%pare$ to gas chro%atograph& or s"percritical fl"i$
e>traction. *n a$$ition+ no che%ical reaction taes place thro"gho"t the process+ lea2ing the
starting %aterial che%icall& "nchange$ after.
The %ain $isa$2antage is the large a%o"nt of ti%e nee$e$ to prepare the col"%n an$
to co%plete the o2erall process. Also+ it %"st be constantl& %onitore$ to ens"re that sol2ent
le2els are replenishe$ an$ the col"%n is !ell:s"pplie$ !ith flo!ing li#"i$+ "nlie !ith paper
chro%atograph& !hich can be left alone $"ring e>peri%entation.
-. References
Ah"Fa S. 0))3. ;hro%atograph& an$ Separation Science. Iol"%e ?. ASAB Aca$e%ic Press.
-raith!aite A+ S%ith FJ. 1(((. ;hro%atographic %etho$s. 5
e$. =or$rechtB Cl"!er
Aca$e%ic P"blishers.
=a2i$ /L. 0))1. Anal&tical ;he%istr&. *n$iaB Ani2ersities Press.
3ar2e& =. 0))). Mo$ern Anal&tical ;he%istr&. ASAB The Mc/ra!:3ill ;o%panies+ *nc.
Cat7 K+ Ksteen R+ Schoen%aers P+ Miller <. 1(('. ;hro%atograph& han$boo of 3PL;.
<e! NorB Marcel =eer+ *nc.
<ielsen S. 0)1). Foo$ Anal&sis. ?
K$. <e! NorB Springer.
Po%eran7 N+ Meloan ;. 0))). Foo$ Anal&sisB Theor& an$ Practice. ASAB Aspen P"blishers+
Raa%an <. 0))8. Ph&toche%ical Techni#"es. <e! =elhi+ *n$iaB <e! *n$ia P"blishing
Sn&$er LR+ Cirlan$ JJ. 1(D3. *ntro$"ction to Mo$ern Li#"i$ ;hro%atograph&+ 0
<e! NorB John Wile& . Sons+ *nc.
Wilson C+ Waler J. 0))). Principles an$ Techhni#"es of Practical -ioche%istr&. 5
ACB Ani2ersit& ;a%bri$ge Press.
Fair J=+ Cor%os ;M. 0))'. Flash col"%n chro%atogra%s esti%ate$ fro% thin:la&er
chro%atograph& $ata. Jo"rnal of ;hro%atograph& A 1011 ,11B ?(:5?.
C"O%iere C+ -al$ K. 0))'. Re$"ce$ an$ total gl"tathione an$ c&steine profiles of citr"s fr"it
F"ices "sing li#"i$ chro%atograph&. Foo$ ;he%istr& 1)8B 3?):3??.
-etanco"rt J+ /ottlieb S. 0)1). Li#"i$ chro%atograph&. =epart%ent of ;he%istr&B Ani2ersit&
of ;alifornia 4 =a2is. P*nternetQ PAccesse$ 0? A"g"st 0)10Q A2ailable fro%B
/albraith Laboratories. 0)11. 3istor& of li#"i$ chro%atograph& testing. /albraith
Laboratories+ *nc. P*nternetQ PAccesse$ 0? A"g"st 0)10Q A2ailable fro%B
Fit7geral$ W. 1((8. ;hro%atographic %etho$s. Science =epart%ent+ Li%eric R.T.;.
Rep"blishe$ in ;he%istr& in ActionS 51. P*nternetQPAccesse$ 0? A"g"st 0)10Q A2ailable
fro%B httpBJJ!!!3."l.ieJTchil$spJ;inAJ*ss"e51Jiss"
Cebbe"s --. 0))D. ;hro%atographic %etho$s. <e! Jerse& *nstit"te of Technolog&B
Kn2iron%ental ;he%ical Anal&sis. P*nternetQ PAccesse$ )1 Septe%ber 0)10Q A2ailable
fro%B httpBJJ!eb.nFit.e$"JTebbe"sJanal&sisJ?;
Ani2ersit& of ;olora$o. 0)10. Organic ;he%istr&B ;ol"%n ;hro%atograph&. =epart%ent of
;he%istr& an$ -ioche%istr&. P*nternetQ PAccesse$ )1 Septe%ber 0)10Q A2ailable fro%
Ani2ersit& of Toronto. 0)1). Thin la&er chro%atograph& theor&. Ani2ersit& of Toronto
Scarboro"ghB ;he%istr& Online. P*nternetQ PAccesse$ 0D A"g"st 0)10Q A2ailable fro%B
Laboratory Manuals
A7ares /F+ -ahra%i ;L+ Me$ina LR+ Me$ina ML;+ L"et"lio OP+ T"a7on CS. -asic Organic
;he%istr& Laborator& Man"al. 0
se%ester+ 0))5:)8. ;ollege of K$"cation+
Ani2ersit& of Santo To%as.
R"binson CA+ R"binson JF. 0))). Li#"i$ ;hro%atograph&. <e! Jerse&B Prentice 3all Press.
S!a%& PM. 0))'. Laborator& Man"al on -iotechnolog&. <e! =elhi+ *n$iaB Rastogi

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