CHROMATOGRAPHIC TECHNIQUES

Aim: Separation and purification of bio o!ica mo ecu e" from a mi#ture of mo ecu e" and in$e"ti!ation of t%eir propertie"& T%e' are u"ed for t%e "eparation of ( ar!e amount" )"e$era !ram"* ( "ma amount" )micro( or pico!ram* of materia " Se ection of a particu ar c%romato!rap%ic met%od depend" on t%e nature of materia to be i"o ated& Se$era c%romato!rap%ic met%od" can be u"ed "e+uentia ' to comp ete purification& Princip e: In a c%romato!rap%ic "'"tem", t%ere are t-o p%a"e"& .( /( Stainar' p%a"e Mobi e p%a"e Separation i" due to di"tribution of mo ecu e" bet-een t-o p%a"e"& Partition )di"tribution coefficient* 0 i" u"ed to de"cribe t%e di"tribution of an' compound bet-een t-o p%a"e"& ( bet-een t-o immi"cib e i+uid" )t-o "o $ent"* concentration in So $ent A concentration in So $ent 1 ( bet-een t-o p%a"e" : "o id 2 i+uid Stationar' Mobi e

E#amp e: If a "ub"tance 3 %a" a di"tribution coefficient of 4&5 in "i icic acid and 2 ben6ene7 4&5 0 .2/, T%i" mean" t%at 839 in ben6ene i" t-ice of t%at in t%e "i icic acid&

:i"tribution coefficient i" defined for c%romato!rap%ic purpo"e" a": concentration of "ub"tance in mobi e p%a"e concentration of "ub"tance in "tationar' p%a"e

Stationar' p%a"e: ma' be ( "o id ( i+uid ( "o id2 i+uid mi#ture )"emi("o id* ( !e and it i" immobi i6ed& Mobi e p%a"e: ma' be ( i+uid or ( !a"eou" and f o-" t%rou!% "tationar' p%a"e&

E#amp e to c%romato!rap%ic "'"tem" -it% different "tationar' and mobi e p%a"e" Stationar' Ad"orption c%romato!rap%' Paper c%romato!rap%' Ga" c%romato!rap%' Ion(e#c%an!e c%romato!rap%' Ge fi tration c%romato!rap%' Affinit' c%romato!rap%' "o id "emi("o id i+uid "o id re"in "o id re"in "o id re"in Mobi e i+uid i+uid !a"eou" i+uid i+uid i+uid

T%e mobi e and "tationar' p%a"e" are c%o"en accordin! to t%e compound to be "eparated&

Genera Tec%ni+ue" of C%romato!rap%'

T%ere are t%ree form" of c%romato!rap%ic "eparation"& .* Co umn c%romato!rap%': Stationar' p%a"e i" pac;ed into ! a"" or meta co umn"&

/* T%in <a'er C%romato!rap%': Stationar' p%a"e i" t%in ' coated into ! a"" or p a"tic "urface"& Stationar' p%a"e ( t%in a'er ad"orbent ! a""

=* Paper C%romato!rap%': Stationar' p%a"e i" "upported b' t%e ce u o"e fibre" )-it% H/O* of a paper "%eet&

Co umn C%romato!rap%'
A of t%e ma>or t'pe of c%romato!rap%ie" are carried out u"in! co umn": Ad"orption, partition, ion e#c%an!e, "i6e e#c u"ion, affinit', !a", %i!% pre""ure i+uid c%romato!rap%ie"& Co umn": G a"" co umn" are common ' u"ed& Co umn" "%ou d %a$e a "upport for t%e "tationar' p%a"e& u"e of porou" ! a"" p ate rep aceab e n' on net p u! of ! a"" -oo

)1uffer*

A capillary tubing for leading effluent from the column to the UVVisible monitor or collection system

Pac;in! of co umn: Pac;in! of co umn i" carried out b' pourin! of " urr' of t%e "tationar' p%a"e )ad"orbent, re"in or !e * into a co umn& Co umn out et mu"t be c o"ed& Note: T%e re"in mu"t be e+ui ibrated -it% t%e buffer to be u"ed before pac;in!& Po-dered "o id partic e" )re"in* -ere "u"pended or "-e ed in buffer& A a'er of "o $ent "%ou d a -a'" be maintained abo$e t%e co umn "urface ( do not dr' !e & E+ui ibration of co umn: After pac;in! t%e co umn, it i" e+ui ibrated -it% buffer& Samp e app ication: Samp e i" app ied to t%e top of co umn b' (pipette ( capi ar' tubin! )"ma "amp e $o ume* ( peri"ta tic pump ) ar!e "amp e $o ume* 1efore app ication of "amp e ( remo$e "o $ent abo$e t%e re"in b' "uction&

Co umn de$e opment )E ution*: T%e component" of t%e app ied "amp e are "eparated b' continuou" pa""a!e of "uitab e mobi e p%a"e t%rou!% t%e co umn& Rate of f o-: i" ad>u"ted or maintained con"tant b' ( peri"ta tic pump or ( Mariotte f a"; ;eep t%e operatin! pre""ure con"tant

Peri"ta tic pump T'pe" of e ution: ( u"e "in! e "o $ent a" t%e e uant ( Gradient e ution", "tep-i"e or continuou", done b' c%an!in! t%e ? pH ? @ )ionic concentration* ? po arit' E#: t-o "o $ent" %a$in! different @ are mi#ed in t%e correct proportion"&
1ufferA 4&B M CC 1uffer )noCC *

4&B M((

S/
)@* to co umn 4

S.
CC conc&

mi#

If t-o c%amber" %a$e t%e "ame dimen"ion" )"ame $o ume* ca ed inear !radient&

Draction co ection and ana '"i": T%e e uted fraction" are co ected in "eparate tube" u"in! an apparatu" ca ed fraction co ector& Eor;" in time drop or $o ume mode

Dor continuou" monitorin! of e uted fraction", UF(Fi"ib e monitor" are u"ed& Simp e p%otomer" E#: Protein" ab"orb UF i!%t at /B4 nm& 1 A C Ab"orbance at Gma# number of tube" )m of e uant* Recorder

E uant" co ected in "eparate tube" u"in! fraction co ector& T%en eac% tube i" ana '6ed for t%e pre"ence of a de"ired protein& E#: tota bio o!ica function& So find t%at in -%ic% tube" )pea; or fraction* t%e protein under intere"t i" e uted& protein determination and determination of acti$it' of protein due to "pecific

T<C

It i" u"ed for "ma amount of materia "& Preparation: t%in a'er ad"orbent )4&/(4&5 mm or 5 mm* ! a"" or p a"tic ( A " urr' of t%e "tationar' p%a"e in H/O i" app ied and coated to a ! a"", p a"tic or foi p ate a" a uniform t%in a'er& ( a uniform a'er obtained b' u"in! a p ate "preader "tartin! at one end of t%e p ate and mo$in! to t%e ot%er end& (t%ic;ne"" of t%e " urr' )"tationar' p%a"e* ( 4&/5 mm for ana 'tica "eparation" ( 5 mm for preparati$e "eparation" ( T%e"e p ate" are a$ai ab e commercia '& ( T%en p ate" are dried in o$en at .44(./4HC& T%i" proce"" acti$ate" ad"orbant A umina Si ica ad"orbant" Samp e App ication: i* Samp e i" u"ua ' o- ME mo ecu e" )a&acid", nuc eotide", fatt' acid", "u!ar"* "o $ent front ii* Samp e i" app ied to t%e p ate b' u"in! a micropipette or "'rin!e& pipette iii* Samp e i" !enera ' app ied /&4(/&5 cm o
. / = /(/&5 cm

from one ed!e of t%e p ate&

)for ana 'tica "eparation" for preparati$e "eparation" "pot app ication band app ication*

dot" )"pot*

"trea; )band*

:e$e opment of p ate:

Tan; S urr' )"tationar' p%a"e* ! a"" p ate

"amp e buffer or "o $ent mi#ture )mobi e p%a"e*

( Separation done in a ! a"" tan; -%ic% contain" t%e de$e opin! "o $ent )mobi e p%a"e* to a dept% of about .&5 cm& ( Tan; i" co$ered b' a ! a"" p ate for e+ui ibration -it% "o $ent $apor& ( After e+ui ibration, t%in a'er p ate i" p aced $ertica ' in t%e tan; and co$ered a!ain& ( Separation of compound" occur" a" t%e "o $ent" tra$e up t%e p ate& Po ar and non( po ar mo ecu e" are "eparated& Q& Ho-I Princip e of "eparationI ( :e$e opment time depend" on t%e "o $ent" ( .4(J4 minute" ( One of t%e ad$anta!e of T<C, it %a" a "%ort time for "eparation&

T-o dimen"iona T<C Aim: to impro$e re"o ution

:irection of "econd de$e opment Direction of first development

1 A o Ori!in So $ent "'"tem .

K4H rotation

So $ent "'"tem /

: C A o Ori!in

T%e "amp e i" app ied to one corner of a p ate a" a "in! e "pot and p ate de$e oped in one direction in "o $ent "'"tem .& T%en p ate i" remo$ed from t%e tan; and dried& It i" de$e oped b' anot%er "o $ent "'"tem )"'"tem /* in a direction at ri!%t an! e to t%e fir"t de$e opment& T%e "pot 1 i" furt%er "eparated into C and : in "o $ent "'"tem /& Component :etection: Qua itati$e detection met%od": .* Spra'in! t%e p ate -it% 54L H/SOM or /5L H/SOM in Et%ano t%en %eatin! )?* burnin! of compound" and bro-n "pot" appear& "%o-" t%e po"ition of UF ab"orbin! aromatic compound" /* E#amination of t%e p ate under UF i!%t =* Co oured compound" "een -it% na;ed e'e& M* Sub>ect t%e p ate to iodine $apor if un"aturated compound" are to be e#amined& I N C0C C(C N I Oe o5* Spra'in! t%e p ate -it% "pecific co our rea!ent" to de$e op co or& E#7 nin%'drin I/ for amino acid" purp e co or

or f uore"cent compound"& )u"e of UF amp*

J* Autoradio!rap%': u"ed for detection of radioacti$e compound"& Tec%ni+ue detect" t%e "pot" a" dar; area" on 3(ra' fi m"& Quantitati$e :etermination: T%e amount of compound ma' be determined b': i* On(p ate +uantification: Quantification of "pot" "ti on t%e p ate b' ( den"itometer": mea"ure t%e UF or $i"ib e ab"orption of t%e compound& ( radioc%romato!ram: u"ed for radio( abe ed compound"& ii* Off(p ate +uantification: ( It i" carried out b' cuttin! off t%e "pot from t%e p ate )p a"tic*& ( t%en e utin! t%e compound -it% a "uitab e "o $ent&

cut off

Samp e "o $ent to re ea"e t%e compound from ad"orbent

E ution

:etection or +uantitation b' ((((((((((((((((((((((((((((((((((((((((((((((((((((( co orimetr'

Paper C%romato!rap%'
Ce u o"e fibre" of paper act a" "tationar' p%a"e )"upportin! matri#*

ma' be ( po ar -ater ( nonpo ar materia ) i+uid paraffin* mobi e p%a"e: buffer or or!anic "o $ent" Paper :e$e opment: T%ere are t-o tec%ni+ue": ( a"cendin! ( de"cendin! trou!% -it% "o $ent Paper o G a"" tan; Samp e app ication So $ent de"cendin! in bot% ca"e", t%e "o $ent i" p aced in t%e ba"e of ! a"" tan; ( E+ui ibration -it% "o $ent $apor ( In a"cendin! tec%ni+ue: ( one end of t%e paper i" in contact -it% t%e "o $ent in t%e ba"e of tan;& ( T%e "amp e "pot" "%ou d be >u"t abo$e t%e "urface of t%e "o $ent ( Mo$ement of "o $ent i" on ' due to t%e capi ar' action and a" t%e "o $ent mo$e" $ertica ' up t%e paper, "eparation of "amp e i" ac%ie$ed& ( In de"cendin! tec%ni+ue: ( T%e paper i" a o-ed to %an! $ertica ' in tan;& Samp e i" on top of t%e paper and top of t%e paper i" in contact -it% t%e "o $ent in trou!% and -etted& T%e bottom of paper i" not in contact -it% t%e "o $ent in t%e ba"e of t%e tan;&
o

a"cendin!

( Mo$ement of "o $ent do-n-ard" i" due to !ra$it' and capi ar' action, "o a" t%e "o $ent mo$e" do-n t%e paper, "eparation of "amp e i" ac%ie$ed& ( D o- of "o $ent i" fa"ter t%an t%e a"cendin! tec%ni+ue& ( Re"o ution i" o-&

A"cendin! paper c%romato!rap%'

T-o dimen"iona c%romato!rap%' -a" a "o u"ed for paper "'"tem" "imi ar to T<C& Q& E%at i" t%e ba"i" of "eparation b' paper c%romato!rap%'I :etection and Identification: Simi ar to T<C but H/SOM i" not u"ed& It di"inte!rate" t%e paper& Durt%er identification of a compound made on t%e ba"i" of t%e Rf $a ue&
So $ent front

"o ute

"tandard

t%e 1 Rf 0

di"tance

mo$ed

b'

t%e di"tance mo$ed Rf 0 A 2 1

b' "o $ent front A A . / = . / = S S i" con"tant for a particu ar compound under "tandard condition" So $ent: mi#ture of $ariou" i+uid" E#: n(butano 2 acetic acid 2 H/O -it% a ratio of 54 2 .5 2 =5

Rf P .&4