CHROMATOGRAPHYTECHNI QUES

Chromatographyi
sav ersat
il
eandwi del
yusedanal yti
calt
echni quef orseparati
ng
andidenti
fyi
ngcomponentswithinami xt
ure.Itr
eli
esont hedi f
ferenti
alinter
acti
ons
ofsampl e component
s with a stati
onaryphase and a mobi le phase.Var i
ous
chr
omat ographyt
echni
quesexi st,eachwi t
hspeci f
icapplicat
ionsandpr i
nci
ples.
Herearesomeoft hemostcommonl yemployedchromatographyt echniques:

(1.)AFFINITYCHROMATOGRAPHY
Affini
ty chromatography is a speci al
ized chromatographict echni
que used t o
separ at
eandpur if
ybi omolecul esbasedont heirspecif
icint
eracti
onswi t
hal igand
i
mmobi li
zed onasol id suppor t
.Thist echni
quet akesadv antageoft heuni que
bindingaffini
ti
esbetweenat argetmol ecule(e.g.
,apr otei
nornucl ei
caci d)anda
specificli
gand,suchasanant ibody,antigen,enzymesubst r
ate,orreceptor.Here'
s
anov ervi
ewofaf fi
nit
ychr omat ography:

Pri
ncipleofAf fi
nityChr omat ogr aphy :Thepr inci plebehi ndaf fi
nit
ychr omat ogr aphy
i
sthesel ect iveandr eversiblebi ndi ngofat argetmol eculetoani mmobi l
i
zedl igand.
Thebasi cst epsi nvolvedi naf fi
nitychr omatogr aphyar easf ollows:
1.I mmobi l
izati
on:A l igand wi th a high af fi
nit
yf ort he t argetmol ecul ei s
cov alentlyat tachedt oasol i
dsuppor t,t y pi
callyachr omat ographycol umn
mat rix.Commonl igandsi ncl udeant i
bodi es,enzy mes,l ectins,andDNA/ RNA
sequences.
2.Sampl eAppl icati
on:Thesampl econt aining t hemi xtureofbi omol ecul es,
i
ncl udingt het argetmol ecul e, i
sappliedt ot hecol umn.Non- specifi
cal
lybound
mol ecul esar ewashedaway .
3.Speci ficBi nding:Thet argetmol eculesel ecti
velybi ndst ot hei mmobi li
zed
l
igand t hroughspeci fici nt eracti
ons,suchasant i
body -
ant i
genorenzy me-
subst ratebi nding.Thi sbi ndi ngi shighlyspeci f
ic,all
owi ngf orthepur i
f i
cation
oft het argetmol ecul efrom t hemi xture.
4.El ution:Theboundt argetmol eculeissubsequent l
yel utedf rom thecol umnby
changi ngt hecondi ti
ons,suchasal teringpH,i onicst rength,oraddi nga
compet iti
v e binding par t ner .This di sr upt st he speci f
ici nteracti
ons and
releasest hetargetmol ecul e.

Appl i
cati
onsofAf fi
ni tyChr omat ogr aphy :Af finitychr omat ographyisav ersat
il
e
technique wi th numer ous appl i
cations i n bi ochemi stry,mol ecularbiology ,and
biotechnology:
1.Pr otein Pur ifi
cat ion:Af fi
nitychr omat ogr aphyi scommonl yused t o purif
y
speci f
icprot ei
nsf rom compl exmi xtures, suchascel l
lysatesorculturemedi a.
Exampl esi ncludei solatingant i
bodi es, enzy mes, andrecombi nantproteins.
2.Ant i
bodyPur i
ficat i
on:Monocl onalandpol yclonalant ibodiescanbepur i
fi
ed
usingcol umnswi t
hi mmobi l
izedant i
gensorpr ot einA/G.
3.Nucl ei
cAci dPur i
fication:Af fi
nitychr omat ographycanbeusedt oisolateDNA
or RNA f ragment s based on t heir speci fi
c sequences or bi nding to
compl ement arysequences.
4.Enzy meKi neticsSt udi es:Immobi l
izedsubst ratesori nhibi
torsareusedt o
studyenzy meki net i
csandi nteractions.
5.Dr ugDi scov ery:Af f
initychr omat ogr aphyi sempl oyedt oscr eencompounds
forbindingaf f
inityt ospeci fi
cdr ugt ar gets, suchasr eceptorsorenzy mes.
6.Bi otechnol ogyandBi opr ocessing:I tpl aysacr i
ticalroleintheproduct ionof
therapeut i
c pr ot eins and t he remov alofcont aminant sf r
om bi oprocess
streams.

Adv
ant
agesofAf
fi
nit
yChr
omat
ogr
aphy
:
1
  Highspeci fi
cit
yandsel ecti
vi
ty.
 Gent lepurifi
cati
onmet hodthatmai ntainsproteinact
ivi
ty.
 Highy i
eldandpur ityofthetargetmol ecule.
 Ver sati
leandappl icabletovariousbiomol ecules.
Chall
enges:
 Ligand i mmobi l
i
zat i
on can be compl ex and may af fectli
gand bindi
ng
proper t
ies.
 Sp ecifi
cligandsmaynotbeav ailabl
ef orallt
argetmolecules.
 Th etechni quemaynotbesui tabl eforlarge-
scalepuri
fi
cationduetocostand
capaci t
yl i
mi t
ati
ons.

(2.)GasChromat ograph(GC)
GasChr omat ography(GC)i sawidelyusedanalyt
icaltechni
quei nchemi st
ryand
biochemist
ryf ortheseparat
ionandanal
ysi
sofvolat
ileandsemi -
volat
ilecompounds
i
ngasesandl iqui
ds.GCi sbasedonthepri
nci
pleofselecti
veparti
ti
oningofanaly
tes
betweenast ati
onaryphaseandamobi legasphase.Her e'
sanov ervi
ew ofgas
chromatography :

KeyComponent sofaGasChr omat ograph:

1.I njecti
onPor t:Thi siswher ethesampl ei sintr
oducedi ntothesystem.I tcan
beaspl it/spli
tlessi njectororadi r
ectinjecti
onpor t
.
2.Col umn:Thecol umni sal ong,narrowtubepackedwi thast ati
onaryphaseor
coat ed wi t
hast ationaryphase.I tist hehear toft heGC sy stem wher e
separ ationoccur s.
3.Car ri
erGas:Ai nert,high-puri
tygas( ty
picall
yhelium,nitrogen,orhydrogen)is
usedt ocar rythesampl ethrought hecolumn.
4.Det ector :Thedet ectori sr esponsibl
ef ormeasur i
ngt heconcent r
ationof
anal yt
esast heyel utef rom thecol umn.Commondet ectorsincl
udeFl ame
IonizationDet ector( FID),Ther malConduct i
vi
tyDet ector(TCD),andMass
Spect romet er(MS) .
5.Dat aSy st em:Acomput er-baseddat asystem coll
ectsandpr ocessesdetector
signals, gener atingchr omat ogramsandquant it
ativ
eresul t
s.

Worki
ngPr inci pleofGC:
1.Sampl eI ntroduct ion:Asmal lamountoft hesampl e( usuall
yi nthel i
quidor
gasphase)i sinj ectedi ntot heGCsy stem.I nt hei nj
ectionpor t
,thesampl eis
vaporizedandi ntroducedi ntot hecar ri
ergasst ream.
2.Col umnSepar ation:Thecar riergascar ri
est hev aporizedsampl et hr
ought he
chromat ogr aphi ccol umn.I nsidet hecol umn, t
hesampl ecomponent sinteract
witht hest at i
onar yphase.Thedegr eeofi nteractiondependsonf act
or ssuch
asv olatili
ty,pol ar i
ty,andaf fi
nityf orthest ationar yphase.
3.Separ at i
on:Ast hesampl ecomponent st raverset hecol umn,t heypar ti
tion
between t he mobi l
e and st ationar y phases.Thi s par t
it
ioning leads t o
dif
ferent ialmi gr ationr ates,causi ngt hei ndiv i
dual component st oseparate.
4.Det ect ion:Ast hesepar atedanal ytesexi tt hecol umn,t heypasst hr
ought he
detect or.Thedet ectorr ecordst hepr esenceandquant ityofeachcomponent
basedonaphy sical orchemi cal proper tyspeci fi
ct othatcomponent .
5.Dat aAnal ysis:Thedat asy stem pr ocessest hedet ectorsignalstogener atea
chromat ogr am, whi chi sapl otofdet ectorresponsev er
sust i
me.Peaksi nt he
chromat ogr am cor r
espondt oi ndividualanal yt
es,andt heirareasorhei ghts
canbeusedf orquant ifi
cat i
on.

Appl
icat
ionsofGC:GCisusedinawi derangeofappl
icat
ions,
includi
ng:
 E nvir
onment
alanal
ysi
s(e.g.
,detect
ionofpol
lut
antsinairandwater)
2
  Foodandbev erageanalysis(e.g.
,fl
avorandar omaanal ysis)
 Pharmaceut i
cal anal
ysis(e.g.,
drugformulati
onandqual i
tycontr
ol)
 Forensicanalysis(e.
g.,drugtesti
ngandar soninv esti
gati
ons)
 Petrochemical anal
ysi
s( e.g.,
deter
mi nat
ionofhy drocarboncompositi
on)
 Cli
nicalanal
ysis( e.
g.,
anal ysi
sofbloodgasesandv ol
atil
eorgani
ccompounds)

Adv
ant
agesofGC:
 Highseparationeffici
encyandresolut
ion.
 Wideapplicabil
it
yt oabroadrangeofcompounds.
 Qu ant
it
ati
veandqual i
tat
iveanal
ysis.
 Fastanaly
sist i
mes.
 Robustandr eli
able.

Chall
enges:
 Limit
edtovolati
l
eorsemi-vol
ati
l
ecompounds.
 Requir
esspecial
izedt
rai
ningandmai
ntenance.
 Safet
yprecauti
onsarenecessar
ywhenhandli
ngsomesampl
esandgases.

(3.)Li
quidChr omatogr
aphy( LC)
Liqui
d Chr omat
ography ( LC)i sav ersati
l
e and wi dely used analyti
cal
technique for the separati
on,i denti
fi
cati
on,and quant i
fi
cation of chemi cal
compoundsi nvari
oussamples.LCi sbasedont hepri
ncipl
eofselecti
vedistr
ibuti
on
ofanal yt
esbet weenamobi leliqui
dphase( theeluentorsolvent)andast ationary
phase( typi
call
yapackedcolumnorasol idsuppor t
).Thi
sseparationisachi
ev edby
exploi
tingdiff
erencesi
nt hei nt
eract
ionsofanal yt
eswi t
ht hestati
onaryphaseand
theeluent.Here'
sanoverviewofl i
qui
dchr omatography:

KeyComponent sofaLi quidChr omat ograph:

1.Pump:Thepumpi sresponsiblef ordeli
veri
ngt hel i
quideluentataconst ant
andcont rolledflowr atethrought hechromat ographicsystem.
2.I njector:Thei nject orisusedt ointroducethesampl eintotheliqui
dst ream.
Commoni njecti
ont echniquesi ncludemanuali njecti
on,autosampl ers,and
onl i
nesampl epr eparation.
3.Col umn:Thecol umni swher et hesepar ati
onofanal ytestakespl ace.I t
cont ainst hest ationaryphase,whi chcanbemadeofv ar
iousmat er
ials,such
assi li
ca, poly mer ,orbondedphases.
4.Det ector:Thedet ectormeasur est heconcent r
ati
onofanal y
tesast heyel ute
from t hecol umn.Ther ear ev ari
oust ypesofdet ectorsusedi nLC,i ncludi ng
UV- Visible,fluorescence, massspect rometry,andrefracti
veindexdetect ors.
5.Dat aSy st em:Acomput er-
baseddat asystem coll
ectsandpr ocessesdet ect or
signals, gener ati
ngchr omat ogramsandquant it
ati
ver esul
ts.

Worki
ngPr incipl eofLC:
1.Sampl eI ntroducti
on:Asmal lamountoft hesampl e( usuall
ydi ssol
vedi na
solv ent)i si nj
ectedi nt othel i
quidchr omat ogr aph.Thesampl eiscarriedby
thel iquidel uentintot hechr omat ographi csystem.
2.Col umn Separ ati
on:I nside t he col umn,t he anal ytes interactwi t
ht he
stationar yphase.Thesei nt eractionscanbebasedonf actorssuchaspol ar
ity,
size,char ge,andaf finit
yf ort hest ationaryphase.Anal yt
eswi t
hdi f
ferent
proper t
ieswi l
lhavedi fferentr et
ent i
ont imesi nt hecolumn.
3.Separ ation:Ast heel uentf l
owst hrought hecol umn,anal yt
esar esepar ated
based on t heirinter actions wi t
ht he st ationary phase.Thi s dif
ferential
i
nt eractioncausest heanal ytest oexitthecol umnatdi f
ferenttimes.
3
 4.Detection:Asanal y
tesexi
tthecolumn,t heypasst hroughthedetector.The
detectormeasur estheconcentr
ati
onofanal ytesandgener atessignal
sthat
arerecor dedbyt hedatasyst
em.
5.DataAnal ysis:Thedatasyst
em processest hedet ect
orsignal
st ogeneratea
chromat ogr am,whichisagr aphi
calrepresent at
ionofanalyteconcentrati
on
overtime.Peaksi nthechromatogram correspondt oindivi
dualanalyt
es,and
thei
rar easorhei ght
scanbeusedf orquant i
ficati
on.

Appl
icat
ionsofLC:Li quidchromat ographyisusedi nv ari
ousappli
cat
ions,i
ncl
uding:
 Ph armaceuti
cal anal
ysis(e.g.,drugpurit
yandf ormulat
ion)
 E nv i
ronmentalanalysi
s( e.g.,detecti
ngpollut
ant sinwaterandair
)
 F oodandbev er
ageanal ysis( e.g.,
ident
if
yingaddi ti
vesandcontaminant
s)
 Cl i
nicalanal
ysis( e.
g.,
anal yzingbloodandur inesampl es)
 Bi otechnol
ogy( e.g.
,analyzingbi omolecul
esl i
kepr otei
nsandnuclei
cacids)
 Pe tr
ochemicalanalysi
s( e.g.,determini
nghy drocarboncompositi
on)

Adv
ant
agesofLC:
 Applicabletoawi der angeofcompounds,i ncludi
ngnonv
olat
il
eandpol
ar
analytes.
 Highsepar ati
oneff
ici
encyandsensi
ti
vi
ty.
 Qu anti
tati
veandquali
tati
veanal
ysi
s.
 Versatil
it
yintermsofcolumnanddetectorchoices.
 Compat i
bil
i
tywithvar
ioussampl
emat r
ices.

Chall
enges:
 Longeranaly
sistimescomparedtogaschromatography
.
 Propermaintenanceandcol
umncar eareessent
ial
.
 Requir
essolventhandli
nganddisposal
consider
ati
ons.

(4.
)COLUMNCHROMATOGRAPHY
Column chromat ography is a wi
dely used chr
omatographict echni
que forthe
separati
on and puri
f i
cat
ion ofchemicalcompounds based on t hei
rdiff
erent
ial
i
nteracti
onswi t
hast ati
onaryphaseandamobi lephaseast heyf l
ow thr
ougha
vert
icalcol
umn.Thi stechniqueisessenti
ali
nchemistry
,bi ochemi str
y,andvari
ous
sci
entifi
cdi
scipl
ines.Here'sanover
viewofcolumnchromat ography :

KeyComponent sofCol umnChr omat ogr aphy :

1.Col umn:Thecol umni sty pi
cal l
yagl assorpl astictubef il
ledwi t
hast at
ionary
phase,suchassi l
i
cageloral umi na.Thechoi ceofst ationaryphasedepends
ont henat ureoft hecompoundst obesepar ated.
2.Sampl eLoadi ngPor t:Thisi sthet opoft hecol umnwher ethesampl emi xt
ure
i
si ntroduced.I tcanbeasi mpl ehol eoraspeci ali
zedinlet.
3.El uentReser voir:Theel uent ,whichi sthesol ventormi xtureofsolventsthat
carriesthesampl ethrought hecol umn, isstoredinanel uentr eserv
oir.
4.Col lect i
onFr actions:Ast heel uentf lowst hrought hecol umnandcar ri
est he
separ ated compounds wi thi t,f ractions oft he eluentar e coll
ected in
cont ai
ner s, oft
eni ntesttubesorv ials.
5.Det ector( Optional):A det ectormaybepl aced att hecol umn'sout letto
moni tort heel uentf ort hepr esenceofcompoundsofi nterest
.Common
detect orsincludeUV- Visibledet ector sandr efract
iveindexdet ectors.

Worki
ngPrinci
pleofColumnChromatography:
1.Sampl eAppl i
cati
on:Thesamplemi xt
ure,di
ssol
vedi
nasuitabl
esolv
ent
,is
car
efull
yint
roducedint
othetopofthecolumn.Thesampl
eisadsor
bedonto
4
 thest ationar yphase.
2.Elution:El uent( asol ventorsol ventmi xture)isaddedt ot hecol umn,whi ch
car r
iest hesampl edownt hrought hecol umn.Ast heel uentf l
owst hroughthe
column,di ff
erentcompounds i nt he sampl ei nter actdi fferentl
ywi t
ht he
stationar yphase.Compounds wi th st
rongeri nter act i
ons wi llmov e more
slowl ythrought hecol umn.
3.Separ ation:Ast heel uentcont inuest ofl
ow, thecompoundswi thinthesampl e
mi xture separ atei nto distinctbands orzones al ong t he col umn.Thi s
separ ationi sbasedonf actorsl i
ket hecompounds' sol ubi
lityintheel uentand
theiraf f
inityforthest ati
onaryphase.
4.Col l
ect i
on ofFr actions:Fr actionsoft heel uentcont aining thesepar ated
compoundsar ecollectedincont ainersastheyexi tthecol umn.Eachf racti
on
maycont ainoneormor ecompounds.
5.Anal ysis:The col l
ect ed fract i
ons can be f urtheranal yzed,of t
en using
spect roscopi coranal yti
caltechni ques,toidentif
yandquant i
fythesepar ated
compounds.

Appli
cat
ionsofCol umnChr omat ogr aphy:Columnchr omatogr
aphyi swi
delyusedin
var
iousapplicati
ons,i
ncluding:
 Pu ri
fi
cation and isolation ofnat uralpr oducts,organic compounds,and
biomol ecul
es(e.g.
,pr oteins,nucleicaci
ds).
 Se parati
on of chemi calr eacti
on pr oducts and i mpuri
ties i
n chemi cal
synthesis.
 An aly
sisofcompl exmi xturesinresearchandqual i
tycont
rollabor
ator
ies.
 Sa mple preparati
on f orsubsequentanal yt
icaltechni
ques such as mass
spectromet r
yornucl earmagnet i
cresonance( NMR)spect r
oscopy.

Adv
ant
agesofCol umnChr omat ography :
 Relativ
elysimpl
eandcost -ef
fecti
v etechni
que.
 Capabl eofseparat
ingawi derangeofcompounds.
 Suitableforbot
hanal y
t i
calandpr eparati
veappl
icat
ions.
 Easilyscaledupforlargersampl equant i
ti
es.

Chall
enges:
 Limitedresol
uti
onforcomplexmi xt
ures.
 Slowercompar edt
omoder nhigh-perf
ormancechr
omatogr
aphi
cmethods.
 Se l
ecti
onofanappropri
atestati
onaryphaseandel
uenti
scruci
alf
orsuccess.

(5.
)PAPERCHROMATOGRAPHY
Paperchromat ographyi sawi delyusedseparati
ont echniquet hatempl oysasheet
ofspecialpaperast hest at
ionaryphaseandal iquidsolventast hemobi lephaset o
separat
e and anal yze the components ofa mi xtur
e.I tis a cost-effecti
ve and
rel
ati
vel
ysi mplemet hodthatiscommonl yusedi nchemi stry
,bi ochemistry,andthe
analy
sisofv ar
ioussubst ances.Here'
sanov er
viewofpaperchr omat ography :

KeyComponent sofPaperChr omatography:

1.PaperSt rip:Ast r
ipoff i
lt
erpaperorchr omatographypaperser v
esast he
stat
ionaryphase.Thi spaperi st ypi
cal
lycutintoal ong,nar row str
ipora
ci
rculardisc.
2.Sampl eSpot :Asmal lspotofthesampl et obeanal yzedi sappliednearthe
baseoft hepaperst r
ip usi
ng acapi l
lar
yt ubeormi cropipett
e.Thespot
containsthemixtureofcompoundst obeseparated.
3.Dev elopingSol vent:A li
quidsolvent,knownast hedev elopingsolvent
,is
pl
acedi nacont ainerorchamber .Thepaperstri
pispar tiall
yimmer sedinthe
5
 sol
v ent ,
all
owi ngthesolventt
otravelupthepapervi
acapi l
laryaction.
4.Cont ainer/Chamber :Thecontai
nerorchamberhol dsthedev elopingsol
vent
andt hepaperst ri
p.Itshouldbet i
ghtl
ysealedtopr ev entt heescapeof
sol
v entv apors.
5.Detect i
on:Af terseparat
ion,thecompoundsar eoftenv isualizedbyv ar
ious
detect i
onmet hods,suchasul travi
olet(UV)li
ght
,iodinev apor,orspecifi
c
chemi cal r
eagentsthatreactwit
ht hecompoundsofinterest.

Worki
ngPr i
ncipleofPaperChr omat ography :
1.Sampl eAppl i
cation:Asmal lamountoft hesampl emi xtureisappl i
edasa
spotneart hebaseoft hepaperst r
ip.Thesampl espotshoul dbewel labov e
thesol ventl evelt
oav oi
ddi r
ectcont actwiththesol v
ent.
2.El ution:Thepaperst ri
pispl acedi nacont ainerorchambercont ainingt he
devel opingsol vent.Ast hesol ventisabsor bedbyt hepaperduet ocapi l
lary
action, i
tmov esupt hest r
ip,carryi
ngt hesampl ecomponent swi thit
.
3.Separ ation:Ast hesol ventt r
av el
st hrought hepaper ,differentcompounds
withint hesampl einteractdi f
ferent
lywi ththepaper 'sfibersandt hesol vent.
Compoundswi t
hst rongeraf fi
nityforthepaperwi llmov emor eslowl ythan
thosewi thweakeraf fi
nit
y,leadingtosepar ati
on.
4.Vi sual i
zation:Af t
erasui t
ablet ime,thepaperi sremov edf rom thechamber ,
and t hesepar ated compoundsar ev i
suali
zed using appr opr i
atedet ecti
on
met hods.Thi s allows f or the ident i
fi
cation and quant if
icati
on of t he
component s.

Appli
cati
onsofPaperChr omat ography :Paperchr omatographyisusedinv ar
ious
appl
icat
ions, i
ncl
udi
ng:
 Se parati
onandidentif
icati
onofami noacids, sugar
s,andorgani
cacids.
 An alysi
sofpigmentsinpl ants,suchaschl orophyll
.
 Qu ali
tati
veanaly
sisofi norganicionsinwat erandsoilsamples.
 Se parati
onofdyesint hetext i
l
eindustry.
 For ensi
canalysi
s,suchasi denti
fyi
nginkcomponent sandfooddyes.

Adv
ant
agesofPaperChr omatography :
 Simpleandinexpensivemet hod.
 Suit
ableforseparati
ngawi der angeofcompounds.
 Canbeusedf orbothquali
tati
veandquant i
tativ
eanalysi
s.
 Minimalequipmentandt echnicalexper
ti
ser equir
ed.

Chall
enges:
 Limit
edresoluti
onforcomplexmixtur
es.
 Result
scanbeaf f
ectedbyvar
iati
onsinhumidi
tyandt
emperatur
e.
 No taseffi
cientasmoder nhigh-
perf
ormancechromatogr
aphicmet
hodsf
or
quanti
tat
iveanalysi
s.

(6.)THI N-LAYERCHROMATOGRAPHY( TLC)

Thin-Lay erChr omat ography(TLC)i sachr omatographictechniqueused f ort he
separ ati
on,i denti
fi
cation,andquantif
icati
onofchemi calcompoundsi nami xture.It
i
ssi mi l
ari npr i
ncipl
et opaperchr omat ographybutempl oysat hinlayerofsol id
adsor bentmat eri
al(usuall
ysil
i
cageloral umina)coat edonaglass, metal
,orpl astic
plateast hest ati
onaryphase,andal iqui
dsol v ent(mobil
ephase)t omov et he
sampl ecomponent salongthest ati
onaryphase.TLC i swidelyusedi nanal y t
ical
chemi stry,biochemi st
ry,andvari
oussci enti
ficfi
elds.Here'
sanov ervi
ewoft hin-l
ay er
chromat ogr aphy :

6
KeyComponent sofThi n- LayerChr omatogr aphy :
1.TLCPl ate:Thest ationaryphasei sat hinlayerofadsorbentmat er
ial(t
y pi
call
y
sili
cageloral umi na)adher edtoaf latsuppor t(usuall
yglassorpl ast
ic)asa
solidpl ate.
2.Sampl eAppl icat ion:Asmal lspotoft hesampl etobeanal yzedi sappli
ednear
thebot tom oft heTLC pl ateusi ngacapi ll
arytube,mi cropi
pette,orot her
suitablemet hod.
3.Dev elopingSol vent :A l i
qui dsolv entorsol ventmi xt
ure( mobi l
ephase)i s
placed i n a chamberort ank.The TLC pl at
ei s positi
oned v erti
call
yor
horizont all
ysot hati tsloweredgei si mmer sedinthesolvent.
4.Cont ainer /
Chamber :Thecont ainerorchamberhol dst hedev elopingsolvent
andt heTLCpl ate.I tisseal edt omai ntai
nasat uratedatmospher ewi thinthe
chamber .
5.Det ection:Af tersepar ation,t he compounds on t he TLC pl ate are often
visualizedusi ngv ar i
ousdet ecti
onmet hods,suchasUVl i
ght ,f
luorescence,or
chemi cal reagent st hatreactwi thspeci f
iccompounds.

Worki
ngPr incipleofThi n-LayerChr omat ography:
1.Sampl eAppl ication:Asmal lamountoft hesampl emi xtureisappliedasa
spotneart hebot t
om oft heTLC pl ate.Thisspotcont ainsthemi xt ur
eof
compoundst obesepar at ed.
2.El uti
on:TheTLC pl atei spl acedi nacont ainerorchambercont ainingt he
developi ngsol v
ent .Ast hesol ventmov esupt hepl atethroughcapill
aryact ion,
i
tcar ri
est hesampl ecomponent swi t
hit.
3.Separ ation:Ast hesol ventt ravelsupt hepl ate,diff
erentcompoundswi thin
thesampl ei nt
eractdi fferentlywi t
ht hest ationaryphaseandt hesol vent.
Compoundswi thst rongeraf fi
nityfort hestat i
onaryphasewi llmov emor e
slowlyt hant hosewi thweakeraf fini
ty,l
eadingt osepar at
ion.
4.Vi sualization:Af tera sui tablet i
me,t he TLC pl at
ei sr emoved f
r om t he
chamber ,and t hesepar ated compoundsar ev isual
ized using appr opriate
detectionmet hods.Thi sal l
owsf ortheidentifi
cationandquant i
fi
cati
onoft he
component s.

Appli
cat
ionsofThi n-LayerChr omat ography:Thin-
layerchromat ographyisusedin
var
iousappli
cat i
ons,incl
uding:
 Qu alit
ativeanalysisofor ganicandi nor
ganiccompounds.
 Pu rit
ytest i
ngofdr ugs,phar maceutical
s,andnat ur
alproducts.
 Mo ni
toringchemi calreactionsandassessi ngreacti
onpr ogress.
 De tecti
on of i mpur i
ties and cont aminants in f ood, bev er
ages, and
envir
onment alsamples.
 F orensicanal y
sis,suchasi dentif
yingdrugsandexpl osiv
es.

Adv
ant
agesofThi n-
LayerChr omatography:
 Quickandcost-ef
fectivemethod.
 Sui
tableforawider angeofcompounds.
 Requir
esmi ni
mal equipmentandt echni
calexper
ti
se.
 Multi
plesamplescanbeanal yzedsi mul
taneousl
yonasi
ngl
eTLCpl
ate.

Chall
enges:
 Limitedresoluti
onf orcomplexmixtur
es.
 Resultscanbeaf f
ectedbyv ar
iat
ionsinhumidi
tyandtemper
ature.
 Qu anti
tati
veanal ysismaybel esspr eci
secompared t
o high-per
for
mance
chromat ographi
cmet hods.
7
(
7.)ION- EXCHANGECHROMATOGRAPHY
I
on-ExchangeChr omat ography(IEC)isapower fulchromatogr
aphictechniqueused
f
ort hesepar at
ionandpur i
fi
cationofchargedi onsandpol armoleculesbasedon
t
heirinteracti
onwi thchargedst ati
onaryphases.Itiswidel
yappli
edinv ar
iousfiel
ds,
i
ncludingbi ochemistr
y,biotechnology,andchemi calanal
ysi
s.Here'
sanov erv
iewof
i
on-exchangechr omatography:

KeyComponent sofI on- ExchangeChr omat ogr aphy :

1.Col umn:Thecol umni spackedwi thast ationaryphase,whi chi st ypicallya
resi norgelcont aini ngchar gedf unct i
onalgr oups.Thechoi ceofst ationar y
phasedependsont hespeci ficionsormol ecul est obesepar at ed.
2.Sampl e Appl icat i
on:The sampl e mi xtur ei si ntroduced i nt ot he col umn
thr ought het op( inject ionpor t).Thesampl econt ainsi onsormol eculeswi th
differ entchar ges.
3.El uent :Theel uent ,al soknownast hemobi lephase,i sabuf fersol uti
ont hat
flowst hr ought hecol umnandhel pst oel ut ethesepar atedspeci es.
4.Det ect or :A det ect ori s used t o moni tort he ef fluentf rom t he col umn,
pr ov iding i nfor mat ion aboutt he el ution t i
mes and concent ration oft he
separ atedcomponent s.Commondet ect orsi ncludeconduct i
vitydet ect ors
andUV- Visibledet ect or s.
Worki
ngPr inci pleofI on-ExchangeChr omat ogr aphy :
1.Sampl eAppl icat i
on:Thesampl ei sloadedont ot het opoft hecol umn, andt he
separ ationbegi ns.
2.I onExchange:Wi thint hecol umn' sst ationar yphase,i onsorpol armol ecul es
i
nt hesampl ei nteractwi t
ht hechar gedf unct i
onalgr oupsont her esinorgel .
Thei nt eract ionscanbeei theri oni cat t
ract ionorr epulsion,dependi ngont he
char gesoft hest ationar yphaseandt hesampl ecomponent s.
3.El ut ion:Theel uenti scont inuousl ypassedt hrought hecol umn.Asi tf lows
thr ought hest at i
onar yphase,i tcompet eswi tht hesampl ei onsf orbi ndi ng
sitesont her esinorgel .Weakl yboundi onsormol ecul esar edi splacedf irst
,
whi l
est rongl yboundonesar eel utedl ater.
4.Separ at ion:Di fferenti onsormol eculesar eel utedatdi f
f er
entt i
mes, r
esul ting
i
nt hei rsepar at i
onbasedont hei rchar gepr oper ties.Thi ssepar ational lows
fort hei solat i
onandpur i
fi
cat ionofspeci fi
ci onsormol ecules.
5.Det ect ion:Theef fluentf rom t hecol umni smoni toredbyadet ector,whi ch
pr ov ides i nfor mat ion aboutt he el ut i
on t i
mes and concent r
ations oft he
separ atedspeci es.

Appli
cati
onsofI on-ExchangeChr omatogr aphy:Ion-exchangechr omat
ogr
aphyi s
usedinvariousappl icati
ons, i
ncludi
ng:
 Se parat i
onandpur ifi
cati
onofproteins,pepti
des,andnucl ei
caci
dsbasedon
theirchar gepr opert
ies.
 An alysisofi norganici onsinenvi
ronment alsampl es,suchaswaterquali
ty
testing.
 Se parat i
onofami noacidsandothersmal lpolarmol ecules.
 Re mov alofi
mpur it
iesandcontami nantsfrom biologicalandphar
maceuti
cal
sampl es.

Adv
ant
agesofI on-ExchangeChr omatography:
 Excell
entforseparati
ngchar gedionsandpol
armolecul
es.
 Highresolut
ionandsel ecti
vi
tybasedonchargeproper
ti
es.
 Versati
leandwi del
yappl i
cabletovari
ouscompounds.

8
Chall
enges:
 Canbesensi t
ivetochangesinpHandi onicst
rength.
 Ma yrequir
ecarefulopt
imizat
ionofbuf
fercondit
ionsforopt
imal
separ
ati
on.
 Limit
edt omoleculeswit
hchar gepr
opert
ies.

(8.)SIZE-EXCLUSI ONCHROMATOGRAPHY
Size-ExclusionChromat ogr
aphy(SEC) ,alsoknownasGelFi l
tr
ati
onChromat ography
orGelPer meati
onChr omatography ,isachr omat
ographict echni
queusedf ort he
separ at
ionandpur if
icati
onofmol eculesbasedont hei
rsizeandmol ecularweight.I
t
i
swi del
yusedi nvariousfi
elds,i
ncludingbiochemistr
y,biotechnol
ogy,andpol ymer
chemi stry.Here'
sanov ervi
ewofsi ze-excl
usi
onchromat ography:

KeyComponent sofSi ze- Excl usionChromat ogr aphy :

1.Col umn:Thecol umnusedi nSECi spackedwi t
hapor ousst ati
onaryphase
composedofcr oss- l
inkedpol ymerbeads.Thesebeadshav easpeci fi
cpor e
sizedi str
ibution, whi chal lowsmolecul esofdi f
ferentsizest oent erorexclude
from thepor es.
2.Sampl eAppl ication:Thesampl emi xturei sinjected ont ot het op oft he
column.Unl ikeot herchr omatographicmet hods,SECdoesnotr equir
epr i
or
sampl epr epar ationori nteract
ionwithasol i
dphase.
3.Mobi lePhase:Themobi lephase( eluent )ist ypicall
yabuf f ersoluti
onor
solventt hatf l
ows t hrough t he column.I tsr olei st o carryt he sampl e
mol eculest hrought hepor ousstat
ionar yphase.
4.Det ect or:A det ect ori s used to moni tort he effluentf rom t he column,
providing inf ormat ion aboutt he elution times and concent rati
ons oft he
separ atedcomponent s.Commondet ect orsincludeUV- Visi
bledet ect
orsand
refr
act iveindexdet ect ors.

Worki
ngPr i
nci pleofSi ze- ExclusionChr omat ography :
1.Sampl eAppl icat i
on:Thesampl eisloadedont ot het opoft hecol umn, andthe
separ ationbegi ns.
2.Por e Si ze Excl usi on:Ast he sampl emol eculesf low through t hepor ous
stationar yphase,t heycanei therentert hepor esorby passt hem basedon
theirsi ze.Smal lermol ecul escanpenet r
atet hepor esandt akel ongerpaths
throught hecol umn,whi lel argermol eculescannotent erthepor esandt ake
shor terpat hs.
3.El ution:Theel uenti scont i
nuousl ypassedt hrought hecol umn.Mol ecul
es
thatent erthepor esspendmor etimewi thinthepor ousmat rix,resulti
ngi na
l
ongerel uti
ont ime.Conv ersel y,moleculesthatcannotent ert hepor estravel
mor equi cklyt hrought hecol umn.
4.Separ ati
on:Ther esulti sasepar ationofmol eculesbasedont heirsize.
Smal l
ermol ecul es el utel at er,whi l
el argermol ecules elut e ear li
er.Thi s
separ ation al lowsf ort hei solati
on and pur if
ication ofspeci fi
cmol ecular
wei ghtr anges.
5.Det ect i
on:Theef fluentf rom t hecolumni smoni t
or edbyadet ector,which
prov i
des i nfor mat i
on aboutt he elution ti
mes and concent r ati
ons oft he
separ atedspeci es.

Appli
cat
ionsofSi ze-Exclusi
onChr omat
ography:Size-exclusi
onchromatogr
aphyis
usedinvari
ousapplicati
ons,includi
ng:
 Separat
ionandpur i
fi
cat i
onofprotei
ns,nucleicaci ds,andpoly
sacchari
des
basedont heirmolecularweight.
 Analysi
sofpol ymermol ecul
arwei
ghtdistr
ibuti
ons.
 Determinat
ionoft hehy drodynami
cradiusofbiomol ecules.
9
  Remov
alofaggr
egat
esandi
mpur
it
iesf
rom bi
ologi
calandphar
maceut
ical
sampl
es.

Adv
ant
agesofSi ze-Excl
usionChromat ogr
aphy
:
 No n-destr
uctivetechniquethatdoesnotr equi
resampleint
eract
ionwi
tha
solidphase.
 Simpl eandr obustmethodsuitablef
orawiderangeofcompounds.
 Highr esol
utionandaccur acyf
ordetermi
ningmolecul
arwei
ghts.

Chall
enges:
 Limitedtothesepar
ati
onofmolecul
esbasedonsi
ze;
otherpr
oper
ti
esar
enot
considered.
 No tsui
tablefort
heanal
ysi
sofsmallmol
ecul
es.

(9.
)HIGHPERFORMANCELI QUI D( HPLC)
High-
Perfor
manceLi qui
dChr omatogr aphy(HPLC)i sapower f
ulanal yt
icalt
echni
que
usedinchemi st
ryandbi ochemi strylaborator
iest osepar at
e,identif
y,andquantify
componentsofcompl exmi xtures.Itisahi ghl
yv ersatil
eandpr ecisemet hodwidely
employedinv ar
iousfi
elds,includingpharmaceut icals,foodanalysis,envir
onmental
monitor
ing,andmedicalbiochemi stry
.Her e'
sanov ervi
ewofHPLC:

Pri
ncipl
eofHPLC:HPLCi sbasedonthepri
nci
plesofl
iqui
dchromatogr
aphy,wher e
asampl ei sdissol
v edi nal i
qui
d(mobil
ephase)andpassedt hr
oughacol umn
packedwi t
hast at
ionaryphase.Thediff
erentcomponentsinthesampleinteract
dif
fer
entlywiththest at
ionar
yphase,l
eadi
ngtoseparat
ionbasedonthei
raff
ini
tyf or
thestat
ionaryphase.

KeyComponent sofanHPLCSy stem:

1.Pump:Thepumpdel iv ersthemobi lephaseataconst antf l
ow rat
et hr
ough
thesy stem.
2.I njector:Theinj
ectorintroducest hesampleint othemobil
ephasest ream.
3.Col umn:Thecol umncont ainsthestati
onaryphaseandi swher eseparati
on
occur s.
4.Det ector:Thedetectormoni torstheeluentleavingthecolumnandpr ovi
des
signalsf ordat
aanalysis.
5.Dat aSy stem:Acomput erordat asystem col l
ectsandpr ocessesdetect
or
signals,generat
ingchr omat ogramsandr esul
t s.

Ty
pesofHPLC:Ther eareseveralvari
ati
onsofHPLC, i
ncludi
ng:
 No rmal -PhaseHPLC:St ati
onaryphaseispol ar,andseparati
oni sbasedon
polaritydifferences.
 Re verse-PhaseHPLC:St ati
onaryphaseisnonpol ar
,andseparationisbased
onhy dr ophobi ci
ty.
 Size-Excl usion(GelFil
trati
on)HPLC:Separ at
ioni sbasedonparticlesi
ze.
 Ion-Exchange HPLC:Separ ati
on is based on i oni
cinteract
ions between
sampl ecomponent sandt hestati
onar
yphase.

Appl
icati
onsofHPLC:HPLCi susedi nvari
ousappli
cati
ons,
incl
uding:
1.Phar maceut i
calAnalysis:Quant i
fyi
ngacti
vepharmaceut i
cali
ngredient
sand
impurit
iesindrugs.
2.Env ir
onment al Anal ysi
s: Det ecti
ng pollut
ants and cont ami nant
s i n
envir
onment alsamples.
3.FoodandBev erageAnal ysis:Det
ermini
ngthecomposi t
ionoffoodpr oduct
s,
addit
ives,andcontaminant s.
10
 4.ClinicalChemi str
y:Analyzing bl ood and ur ine sampl es for di
agnosti
c
pur poses,i
ncl
udingdrugmoni toringanddi seasebi
omar kers.
5.Prot eomicsandBi ochemistr
y :Separ ati
ngandquant if
yingbi omol
eculesli
ke
prot ei
ns,nucl
eicaci
ds,andmet aboli
tes.
6.Qual it
yCont rol
:Ensuring productqual i
tyin manufact uri
ng pr
ocesses by
testingrawmat eri
alsandfi
nal product s.

Adv
ant
agesofHPLC:
 Highsensitiv
ityandprecisi
on.
 Broadappl i
cabil
it
ytoawi derangeofcompounds.
 Abilit
ytoseparateandquant i
fycomplexmixt
ures.
 Aut omati
onandhi gh-
throughputcapabi
li
ti
es.
 Qu anti
tat
iveandquali
tativeanal
ysi
s.

Chall
enges:
 Ini
ti
alset
upcost scanbehigh.
 Me t
hoddev el
opmentandopt i
mizati
onmaybetime-consuming.
 Propermaintenanceandcali
brat
ionarecr
uci
alf
orreli
abler
esult
s.

(10.)CHIRALCHROMATOGRAPHY
Chiralchromatographyisaspeci ali
zedfor
m ofchr omatographyusedtosepar ate
enant i
omers,which are mirror
-i
mage isomer s ofchir
almol ecul
es.Chirali
ty,or
handedness,isapr oper
tyofmol eculest
hathav enosuperi
mposablemirrorimages,
l
ikel eftandrighthands.Separatingenant
iomer sisessenti
alinpharmaceut i
cals,
wher eoftenonlyoneenantiomerofadr ugi sbiol
ogi
call
yactiveandsafe,whil
et he
othercanhav eadverseeff
ects.Here'
sanov erv
iewofchir
alchromatogr
aphy :

KeyComponent sofChiralChromat ography :

1.Chi ralSt ati
onaryPhase:I nchi ralchr omatography,
thest ati
onaryphasei nt
he
chromat ographiccol umni smodi fiedtoi ncludechir
almol eculesorchiral
selectors.Thesesel ectorsi nteractselectivel
ywithoneenant i
omerort he
other,all
owi ngfortheirsepar ati
on.
2.Sampl e Appl i
cation: The sampl e, typi
call
y containing a mi xtur
e of
enantiomer s,i
sintroducedi ntot hechr omatographi
ccolumn.
3.Mobi l
ePhase:Themobi l
ephase,asol ventorbuf f
er,car r
iest hesample
component sthrought hecol umn.
4.Det ect or:Adet ector,suchasUV- Visi
bleoraspeci ali
zedchi raldetect
or,i
s
usedt omoni tortheef fluentf rom t hecolumn,pr ov
idingi nf
ormat i
onabout
el
ut i
ont i
mesandconcent r
ations.

Worki
ngPr i
ncipleofChi ralChromat ography :
1.I nteracti
onwi thChi ralSelect or:Ast hesampl epassest hr
oughthechi ral
column,t he chi r
alsel ector si nt he st ationar
y phase int
eractwitht he
enant i
omer ssel ectivel
y.Thisi nteractionmayi nvol
vehydrogenbonding,van
derWaal sforces,orot hermol ecularint eractions.
2.Separ ati
on:Becauseoft hesel ect
ivei nt eracti
ons,oneenanti
omerwi l
lmov e
mor eslowl yt hr
ought hecol umn( retar dation)duet ostr
ongerbindi
ngt othe
chiralselectors,whi l
etheot herenant iomerwi l
lel
utemorerapidl
y.
3.El utionand Det ect i
on:Theenant iomer sar eelutedfrom thecolumnand
detectedbyt hechr omat ogr
aphi cdet ect or.Theenantiomerscanbequantif
ied
basedont heirelutiontimesandconcent r
ations.

Appli
cati
onsofChi
ralChr
omatography
:
Chi
ralchromat
ogr
aphyhasnumerousappl
i
cat
ionsi
nvar
iousf
iel
ds:
11
 1.PharmaceuticalIndust ry:I
tisusedfortheseparati
onofenanti
omer sofdrugs
andphar maceutical
st oensur ethesafetyandeffi
cacyofmedicati
ons.
2.Agrochemicals:Chi ralchr omatogr
aphyi sempl oyedinthesepar ati
onand
anal
ysisofchiralpest i
cidesandherbicides.
3.FoodandFl avorI ndust ry
:Ithelpsinanal yzi
ngchi r
alcompoundsi nfood
addit
ivesandflav or
ings.
4.Envir
onment alMoni toring:Iti
susedf ort heanal
ysisofchi
ralpoll
utantsand
envi
ronmental contami nants.

Adv
ant
agesofChi ralChromatography:
 Preci
sesepar at
ionofenantiomers,cr
uciali
nphar
maceut
ical
s.
 Highselect
ivit
yforchi
ralcompounds.
 Suit
ableforbothanaly
ticalandpreparat
ivepur
poses.

Chall
enges:
 Chiral
chr
omatogr
aphycol
umnsandselect
orscanbeexpensi
ve.
 Me t
hoddevel
opmentandopt
imi
zat
ioncanbetime-
consuming.

(11.
)REVERSED- PHASECHROMATOGRAPHY
Reversed-phasechromatography(RPC)isawidelyusedchromat ographictechnique
i
nwhi chthestati
onaryphaseismor epolart
hant hemobilephase.I tisparti
cularl
y
usefulfortheseparat
ionandanalysi
sofnonpolarandmoder atelypolarcompounds.
RPCi sof tenemployedinanalyti
calchemist
ry,pharmaceut
icals,andbi ochemistry
.
Here'sanov erv
iewofrever
sed-phasechromatography:

KeyComponent sofRev ersed-PhaseChr omat ography:

1.Col umn:Thecol umnusedi nRPCi stypicall
yfi
l
ledwi t
hanonpol arstat
ionary
phaset hatisusual lyhy drophobi c.Commonst ati
onaryphasesi ncl
udeC18,
C8,andC4si li
ca- basedmat eri
als,butot herphaseswi t
hdi f
ferentalkylchain
l
engt hsar ealsoav ail
able.
2.Sampl e Applicat i
on:The sampl e,di ssolv
ed in an appropri
ate solvent,is
appliedt othet opoft hecol umn.
3.Mobi lePhase:Themobi l
ephaseconsi stsofapol arsolv
entorami xtureof
solvents.I tisl esspol art han t he stati
onaryphase,cr eati
ng a r et
ent i
on
mechani sm basedonhy drophobici nteract
ions.
4.Det ect or:Adet ector,suchasUV- Visi
bleoramassspect romet er,isusedt o
moni tort heeffluentf r
om t hecol umnandpr ovi
dei nf
ormationaboutel uti
on
ti
mesandconcent r
ations.

Worki
ngPr incipl
eofRev ersed- PhaseChr omat ography :
1.Sampl eLoading:Thesampl eisloadedont othecolumn.
2.Ret ent i
on Mechani sm:I n RPC,t he r etenti
on mechani sm i s primaril
y
hydr ophobici nt
eractions.Nonpol arormoder atelypolarcompoundsi nt he
sampl ei nt
eractwi tht he hydrophobic st at
ionary phase,l eadi
ng t ot hei
r
retent i
onont hecolumn.
3.El ution:The mobi le phase i s continuousl y passed through the col umn.
Compoundsar eelutedbasedont heirhy drophobici
ty,wit
hl esshy drophobic
compoundsel uti
ngear l
ierandmor ehy drophobiccompoundsel ut
inglat
er .
4.Separ ati
onandDet ection:Thecompoundsar esepar at
edast heyelutef r
om
thecol umnandar edet ectedbyt hechr omat ographicdetector.Thedet ector
prov i
desi nfor
mat i
onaboutel uti
ont i
mesandconcent rat
ions.

Appli
cat
ionsofReversed-PhaseChromat
ogr
aphy:
Rever
sed-phasechr
omat ographyi
susedi
nawiderangeofappl
i
cat
ions,
incl
udi
ng:
12
 1.Pharmaceut icalAnal y si
s:I tisusedf ortheanalysi
sandqual i
tycontr
olof
pharmaceut i
cal drugsanddr ugformulati
ons.
2.Proteomi csand Met abolomi cs:RPC i sempl oyed f
ortheseparati
onand
quant i
fi
cati
onofpept ides, prot
eins,andmet abol
it
es.
3.Envi
r onmentalAnal ysi s:Itisusedt oanalyzepoll
utant
s,pest
ici
des,
andot her
envi
ronment al contami nant s.
4.FoodandBev erageI ndust ry:RPCisappl i
edintheanalysi
soffoodadditi
ves,
fl
avor s,
andcont ami nant s.

Adv
ant
agesofRev er
sed-PhaseChromatography:
 Versati
leandwi del
yappli
cabl
eforvari
oust ypesofcompounds.
 High-r
esoluti
onseparati
onofnonpolarandmoder atel
ypol
arcompounds.
 Compat ibl
ewithav ar
iet
yofdetect
ors,i
ncludingmassspectr
ometr
y.

Chall
enges:
 Ma ynotbesuit
abl
eforhighl
ypolarcompounds.
 Me t
hoddevelopmentandopti
mizati
oncanbecompl
ex.

(12.)HYDROPHOBI CINTERACTI ONCHROMATOGRAPHY

Hy drophobicInteracti
on Chr omat ography( HIC)is a chromatographi
ct echnique
usedf orthesepar ati
onandpur ifi
cationofbi omolecul
es,suchaspr oteinsand
nucleicacids,basedont heirhydrophobi ci
tyorhydrophi
li
city
.Itisav al
uabletoolin
biochemi st
ry,bi
otechnology ,andthephar maceuti
cali
ndustry.Here'
sanov ervi
ewof
hydrophobicinteracti
onchr omatogr aphy :

KeyComponent sofHy dr ophobi cInteract i

onChr omat ogr aphy:
1.St ationaryPhase:Thest ationar yphasei nHI C consi stsofahy drophobic
mat ri
x.Commonl yusedhy drophobi cl i
gandsi ncludeal ky
lchains,suchas
butyl,octyl,
orpheny lgroups,cov alentlyat t
achedt oasol i
dsupportmat er
ial
.
2.Sampl eAppl i
cat ion:Thesampl e,whi chcont ainst hebi omoleculest o be
separ ated,i
sappl iedt ot hecolumn.
3.Mobi lePhase:Themobi lephaset y
picallycont ainsahi ghconcent r
ationofa
chaot ropicsal
t, suchasammoni um sul fateorsodi um chl or
ide,
inanaqueous
solution.Thi s sal tdi sruptst he wat erst ructur e around the hydrophobic
regionsoft hest at ionaryphaseandweakenst hehy drophobicinteracti
ons
betweent hebiomol eculesandt hest ationaryphase.
4.Det ect or:A det ect or,of t
enaUV- Visibledet ector ,i
sused t o moni torthe
effl
uentf r
om t hecol umnandpr ovi
dei nf
ormat i
onaboutel ut
iontimesand
concent rati
ons.

Worki
ngPr incipleofHy dr
ophobi cInteractionChr omat ography :
1.Sampl eLoadi ng:Thesampl eisloadedont ot hecol umni nabuf fercontaining
thechaot ropicsalt.Int hepr esenceoft hechaot r
opicsalt,thehy dr
ophobi c
i
nt er
act i
onsbet weent hebi omol eculesandt hest ati
onaryphasear einit
iall
y
strong.
2.Equi li
br ati
on:Thecol umni sequilibratedwi ththemobi lephasecont ai
ningt he
chaot r
opi csalt.
3.El uti
on:Ast hemobi l
ephasef lowst hr ought hecol umn,theconcent rat
ionof
the chaot ropi
c sal tgr adual
ly decr eases.As a r esult
,t he hy dr
ophobi c
i
nt er
act i
onsbet weent hebi omol eculesandt hest ati
onaryphaseweaken.The
biomol eculesel utefrom t hecol umni nor derofdecr easinghy drophobicity,
withthemosthy drophobi cmolecul esel utinglast .
4.Separ ationandDet ection:Thebi omol ecul esar esepar atedast heyelutefrom
thecol umnandar edet ectedbyt hechr omat ogr aphicdetector.Thedet ector
13
 pr
ovi
desi
nfor
mat
ionaboutel
uti
ont
imesandconcent
rat
ions.

Appli
cationsofHy drophobi cI nteracti
onChr omat ogr aphy:
Hydrophobici nter
act i
onchr omat ographyisusedi nv ar i
ousappl i
cati
ons,incl
uding:
1.Pr oteinPur ifi
cation:I tiswi delyusedf ort hepur ifi
cati
onofpr otei
nsand
enzymes,par t
icularl
y when ot her chromat ographict echniques li
ke i on
exchangechr omat ographyorsi ze-
exclusi
onchr omat ographyarenotsui t
able.
2.Vacci neDev elopment :HI Ci sempl oyedint hepur i
ficati
onofv i
ralantigens
andr ecombi nantpr ot
ei nsf orvacci
nepr oduction.
3.Bi ophar maceut i
cals:Iti susedf orthepur i
ficat i
onofmonocl onalantibodies
andot herbiophar maceut ical pr
oducts.
4.Nucl ei cAci dPur i
ficati
on:HI Ccanbeusedf ort hepurifi
cati
onofpl asmidDNA,
RNA, andol i
gonucl eotides.

Adv
ant
agesofHy drophobicInter
acti
onChromat ography
:
 Eff
ecti
veforsepar ati
ngandpur if
yingprotei
nsandotherbi
omolecul
esbased
onthei
rhy drophobici
ty.
 Sui
tabl
e f or pur i
fying labi
le molecules that may denat
ure in ot
her
chr
omat ographictechniques.

Chall
enges:
 Carefulopti
mizat
ionofsal
tconcent
rati
onandpHisrequi
redforsuccessful
separati
ons.
 Hydrophobicli
gandscaninter
actdif
ferent
lywi
thv
ariousbi
omolecules,so
methoddev el
opmentcanbecomplex.

14