Phoebe Jilliane O.

Lacson FS 135
Anna Margarita P. Tongco Written Report
LIQUID CHROMATOGRAPHY
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
th
;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.
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$
chro%atograph&.
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.
2
="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 $iagra% e>plaining %o2e%ent of co%ponents in a chro%atographic
process.
Th"s+ li#"i$ 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+
1(((1.
3
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.
4
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
f
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
f
2al"es+ i.e.+ retar$ation factors ,Fig.31.
5
Fig"re ?. R
f
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
f
2al"e ,Ah"Fa+ 0))31. The $istance %o2e$ b&
the sol2ent fronts+ A an$ - ,$
1
+ $
0
+ $
3
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
f
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
f
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.
6
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.
7

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$+ 0))11
8
1.3 Applications
*n foo$ 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
Ʌ
is the $istance %o2e$ b& the anal&te fro% the origin an$ w is the
9
!i$th of the spot an$ the capacit& factor+ k’+ for the anal&te+ is gi2en !here d
f
is the $istance
%o2e$ b& the sol2ent front fro% the origin ,Wilson . Waler+ 0)))1.

3o!e2er+ $ifferent co%ponents !ill ha2e $ifferent R
f
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.
**.0*nstr"%entation
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
10
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
0
SO
?
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
11
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 $iff"sion ,R"binson . R"binson+ 0)))1.
A ( B ( C
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOO OOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOO OOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOO OOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
Sa%ple
,AE-E;1
;ol"%n
Soli$ Particles
,pacing %aterial:
stat ionar& phase1
Kl"ant ,el"ate1
DIAGRAM O) *IMPL+LIQUID COLUM, CHROMATOGRAPHY
A
B
C
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 $ifferences in the si7e of the sol"tes. Large sol"tes are "nable to
12
penetrate into the poro"s stationar& phase an$ 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.
13
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.
14
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 $"ring e>peri%entation.
15
-. References
Books
Ah"Fa S. 0))3. ;hro%atograph& an$ Separation Science. Iol"%e ?. ASAB Aca$e%ic Press.
-raith!aite A+ S%ith FJ. 1(((. ;hro%atographic %etho$s. 5
th
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. ?
th
K$. <e! NorB Springer.
Po%eran7 N+ Meloan ;. 0))). Foo$ Anal&sisB Theor& an$ Practice. ASAB Aspen P"blishers+
*nc.
Raa%an <. 0))8. Ph&toche%ical Techni#"es. <e! =elhi+ *n$iaB <e! *n$ia P"blishing
Agenc&.
Sn&$er LR+ Cirlan$ JJ. 1(D3. *ntro$"ction to Mo$ern Li#"i$ ;hro%atograph&+ 0
n$
K$ition.
<e! NorB John Wile& . Sons+ *nc.
Wilson C+ Waler J. 0))). Principles an$ Techhni#"es of Practical -ioche%istr&. 5
th
K$ition.
ACB Ani2ersit& ;a%bri$ge Press.
Journals
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??.
Internet
-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
httpBJJche%!ii."c$a2is.e$"JAnal&ticalR;he%istr&J*nstr"%entalRAnal&sisJLi#"i$R;hro%
atograph&
16
/albraith Laboratories. 0)11. 3istor& of li#"i$ chro%atograph& testing. /albraith
Laboratories+ *nc. P*nternetQ PAccesse$ 0? A"g"st 0)10Q A2ailable fro%B
httpBJJ!!!.galbraith.co%Jli#"i$:chro%atograph&:testing.ht%
Fit7geral$ 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$spJ;inAJ*ss"e51Jiss"e51RfilesJ%ain.ht%
Cebbe"s --. 0))D. ;hro%atographic %etho$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$"JTebbe"sJanal&sisJ?;3ROMAT.ht%
Ani2ersit& of ;olora$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%
httpBJJorgche%.colora$o.e$"JTechni#"eJProce$"resJ;ol"%nchro%J;ol"%nchro%.ht%l
Ani2ersit& of Toronto. 0)1). Thin la&er chro%atograph& theor&. Ani2ersit& of Toronto
Scarboro"ghB ;he%istr& Online. P*nternetQ PAccesse$ 0D A"g"st 0)10Q A2ailable fro%B
httpBJJ!ebapps."tsc."toronto.caJche%istr&onlineJtlc.ht%l
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
n$
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
P"blications.
17

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