PROJECTON

PRACTI
CE SCHOOL
BY
MANI
SHJ
AISWAL
(
1908820500029)
SUBMI
TTEDASREPORT REQUI
REMENT
BACHELOROFPHARMACY
(
201
9-23)
PROJ
ECTGUI
DE
MR.SANDEEPSI
NGH

DR.ABDULKALAM TECHNI
CALUNI
VERSI
TY,
LUCKNOW

ACKNOWLEDGEMENT

Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
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Thi
sthesi
sistheresul
tofthr
eeyearsofwor k,duri
ngwhichseveralpeopl
ehave
pl
ayedacruci
alrol
einitsc
ompleti
on.I
tisagr eatpl
easur
et hatnowIhavet he
oppor
tuni
tytoexpressmygrati
tudetoallofthem.

AboveallIthanktheal
might
yf orgivi
ngmec our
agetoundertakeitasmypr
oject,
thepatienc
ef orcompl
eti
ngitandf oragui
de,sosuppor
tive,wit
houtwhomthis
taskcouldneverhavebeenful
fil
led.

Itookthi
soppor
tunit
yt othankt
heTAHI
RAINSTITUTEOFMEDICALSCI
ENCES
Coll
egeandourmanagersi
rMR.SHOEBAHMADf orgivi
ngmechancetodothis
proj
ect.

Iwoul
dl i
ketothankmypr inci
palDR.UDAIAGRAHARIsi r
,DeanMR.SHIVSHANKAR
PANDEYsir,Vi
cePrinci
palMR.ABHAYM. L.VERMAsir,HeadOfDepartmentMR.
AJAYYADAVsir,Coordi
natorMR.AMI TKUMARRAIsi r
,Proct
orDR.SHAHEENKHAN
mamf orprovi
dingthenecessaryfac
ili
tiesrequi
redforcomplet
ionofthi
sproj
ect
.

Iwoul
dalsoli
ket
oexpressmysincer
egrat
itudet
owar dsmyproj
ec tguideMR.
SANDEEPSI
NGHsirwhosegui
danceandc
aremadet hi
sprojec
tsuccessful.

Iwouldal
soli
ketothankmyf ac
ult
ymembersMR.BRI
JESHSHARMAsir,MR.
ANKITMISHRAsi
r,MRS.SANDHYAYADAVmam,MR.ANUJSRIVASTAVAsi
r.

CERTI
FICATI
ON

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nst
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calSc
ienc
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Thisistoc er
t i
fythatthi
sproj
ecttit
led“Projec
tOnPr act
iceSc
hoolRepor t
”is
submittedinr epor
trequi
rementforthedegreeofBachel
orinPharmacyof
TAHIRAI NSTI
TUTEOFMEDI CALSCI
ENCES,GIDA,GORAKPUR,U. P.byMANI SH
JAI
SWAL,Rol lno.1
908820500029hasbeenexec ut
ed underoursuper
visionand
guidanc
e.

Thedat arepor
tedinitispure.Theassistanc
eandhelpreceivedduri
ngthi
s
i
nvestigat
ionhasbeendulyac knowledge.I
tisfur
thercer
tifiedthati
tisan
ori
ginalpi
eceofwor kconsi
derf orawar dofdegr
eeofBac helori
nPharmacy.

Dr.UdaiChandAgr
ahar
i Mr
.Sandeep
Si
ngh PRINCI
PAL
Proj
ectguide TAHIRAINSTI
TUTEOFMEDI
CALSCI
ENCES
GI
DA,GORAKHPUR

DECLARATI
ON

Tahi
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Ihereby,MANI SHJAI
SWAL,Rol lno.1908820500029,dec l
aredthatIhavedone
thepr oj
ectreportonthet opi
c“Chr omatography”whi chissubmitt
edas
proj
ec trequi
rementforthedegr eeofBac hel
orinPhar macyfromTahira
I
nstituteofMedicalSci
ences,GIDA,Gorakhpur.I
ti sori
ginalworkdonebyme
andt heinfor
mationprovi
dedint hestudyi sauthenti
ct othebestofmy
knowledge.

Thi
sstudyhasnowbeensubmit
tedt
oanyot
heri
nst
itut
ionoruni
ver
sit
yfor
theawardofanyot
herdegree.

Dat
e:
ManishJ
aiswal

Pl
ace:Gor
akhpur

Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
GIDA,Gor
akhpur
(U.
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 I
NDEX

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(U.
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 I
NTRODUCTI
ON

Chr
omat
ogr
aphy:
Chr omatogr aphyr epresentsagr oupofmet hodsf orsepar ati
ngmol ecul
ar
mi xtur
est hatdependont hedi f
ferenti
alaffi
niti
esoft hesol utebetweent wo
immi sc
iblephases.Oneoft hephasesisaf i
xedbedofl argesur faceareawhichis
cal l
edast at i
onar yphasewhi letheotherisaf l
uidorgaswhi chmovest hroughor
overt hesur fac
ei scall
edmobi lephase.Thestationaryphasemaybeapor ousor
finelydividedsol i
dorl i
quidt hathasbeenc oatedast hinlayeronani nert
suppor tmat eri
al.Themobi lephasemaybeapur el i
quidorami xtureofsoluti
ons
ori tmaybeagasormi xtureofgases.

Chromatographyisnondest
ructi
vepr oc
edureforresol
vingamulti-c
omponent
mixtureoftrace,mi
nor,ormajorconsti
tuentsi
ntoitsi
ndivi
dualfracti
on.Whil
e
chromatogr
aphymaybeappl iedbothquantit
ati
vel
y,iti
spr i
maril
yasepar at
ion
tool.

Tahi
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nst
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Howeverf i
nali dentifi
cationusual l
yr equi
resconfi
rmati
onbysomeot her
analyti
calpr ocedur esuc hasi nfr
ar edspectrosc
opy,nuclearmagneticr esonance
ormasss pect r
omet r
y.Chr omat r
ogr aphycanbeusedf ort hequanti
tat i
veand
quali
tativeanal ysis.Essentiall
y,thet echni
queofc hr
omat ography  isbasedon
thedifferenc esint her ateatwhi chc omponentsofmixturemovest hr ougha
porousmedi um(c all
edst ationary  phase)undert heinfluenceofsomesol vent
orgas(c all
edmovi ngphase) .

Chromatographyinvolvesasampl e( orsampleextr act)beingdissolvedin

a mobilephase ( whichmaybeagas,al iquidorasuper cri
tic
alf l
uid).The
mobil
ephasei sthenfor c
edt hroughani mmobi l
e,immiscible st ati
onaryphase.
Thephasesar echosensuc hthatc omponent softhesampl ehavedi fferi
ng
sol
ubil
iti
esineachphase.Ac omponentwhi chisquit esolubleint hestationar
y
phasewillt
akelongert ot r
avelthr oughitthanac omponentwhi chisnotver y
sol
ubleinthestationaryphasebutver ysolubleint hemobi l
ephase.

Hi
stor
yofChr
omat
ogr
aphy
Thest udyofchromatographystar
t edintheeighteenthc
enturywhenRunge
studi
edwi t
hgr eati
nterestthenatureofinorganiccompoundsof i
lterpapers.He
separatedinor
ganicsal
tsandobser vedthati
nor gani
csal
tst r
aveltodifferent
extentproduci
ngat t
racti
vepattern.

Intheyear1898,DayinUSAf orc
ed
crudepetrol
eumt hroughac olumnofl i
mest oneandfull
er’
searth.Heobserved
thatfi
rstpor t
ionwasofl i
ghthydr ocarbons,foll
owedbyhydrocarbonsof
aromati
cnat ure,unsaturatedt ype,heterocycl
icandni
trogen-sul
phurcontaini
ng
highmolec
ularwei ghthydrocarbons.

TheRussi
anbotanist
,MichaelTswet
tdiscover
edt hechr
omat ogr
aphic
pri
nci
plein1906,heusedaglasscolumnofcalc
iumc ar
bonateforseparati
onof
chl
orophyl
lpigmentsfromplantbyusingpet
roleumet her
.Thepigments,

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acc
ordi
ngt otheiradsor
pti
onpat
ter ns,wer
eresol
vdi
ntovar
iousc
olour
edz
ones;
hethensepar
at edandesti
mat
edt hem.

Between1910and1930,veryli
ttleworkwaspublishedaboutc hromatography.
Themaj ordevelopmentocc ur
redaround1930when Leder erandc o-workersin1
931,
separatedlutei
nandxant henesonac olumnofcal
ciumc ar bonat
epowder .
Furtherdevelopmentssoonf oll
owedwhenKuhn,Kar rerandRuz ickaseparated
plantcarotenesintoseveralcomponentsbyadsor
pt i
onc hr omatography.

*Component
sofc
hromat
ogr
aphy
i
)Mobil
ephase:
-Gasorl
iqui
dthatc
arr
iest
hemi
xtur
eofc
omponent
sthr
ough
t
hestati
onar
yphase.

i
i)St
ati
onar
yphase:-Thepartoftheappar
atust
hathol
dst
hec
omponent
sas
t
heymovethroughi
t,separ
atingt
hem.

*UsesofChr
omat
ogr
aphy
Chr
omat
ogr
aphyi
susedbysc
ient
ist
sto:

:
:Anal
yse-exami
neami
xtur
e,i
t’sc
omponent
s,andt
hei
rrel
ati
onst
ooneanot
her

:
:Ident
ify-det
ermi
net
hei
dent
ityofami
xtur
eorc
omponent
sbasedonknown
c
omponent s

::
Puri
fy-separ
atec
omponent
sinor
dert
oisol
ateoneofi
nter
estf
orf
urt
her
study

:
:Quant
ify-det
ermi
net
heamountoft
hemi
xtur
eort
hec
omponent
spr
esenti
n
t
hesampl e

Real
-l
ifeexampl
esofusesf
orc
hromat
ogr
aphy:

:
:Phar
mac
eut
icalc
ompany

:
:Hospi
tal

:
:Lawenf
orc
ement

:
:Envi
ronmentAgenc
y

Tahi
raI
nst
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eofMedi
calSc
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GIDA,Gor
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(U.
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:
:Manuf
act
uri
ngpl
ant

Basi
cChr
omat
ogr
aphi
cTer
minol
ogi
es
* Chr
omat
ogr
aph:
 Inst
rumentempl
oyedf
orac
hromat
ogr
aphy.
* Stati
onaryphase:
 Phaset hatstaysinpl
aceinsidethec ol
umn.Canbea
par
tic
ularsol
idorgel-basedpacki
ng(LC)orahighlyviscousli
qui
dc oat
edont
he
i
nsi
deoft hecol
umn( GC).
*Mobi
lephase:
 Sol
ventmovi
ngt
hrought
hec
olumn,ei
theral
iqui
dinLCorgas
i
nGC.
El
uent
: Fl
uident
eri
ngac
olumn.

El
uat
e: Fl
uidexi
tingt
hec
olumn.
* El
uti
on:
 Thepr
ocessofpas
singt
hemobi
lephaset
hrought
hec
olumn.
*Chr
omat
ogr
am:Gr
aphshowi
ngdet
ect
orr
esponseasaf
unc
tionofat
ime.
* Fl
owr
ate:
 Howmuc
hmobi
lephasepassed/mi
nut
e(ml
/mi
n).

3
* Li
nearvel
oci
ty:
 Di
stanc
epassedbymobi
lephaseper1mi
nint
hec
olumn.

Thec hr
omat ographicmet hodofsepar ation,ingener al
,involvesthef ol
lowing
steps:
1
.      Adsor pti
onorr et entionofasubst anceorsubst anceont he
stati
onaryphase.
2.
      Separ ationort her etenti
onofasubst anc
ebyt hemobi l
ephase.
3.
      Rec over yoft heads orbedsubst ancesbyac onti
nuousf l
owoft he
mobilephase;themet hodbei ngc all
edelution.
4.
      Qual itati
veandquant i
tat
iveanal ysisoftheel utedsubstances.

*TYPESOFCHROMATOGRAPHY
I
nal ltypesofc hromatography,separati
onoft hecomponent sofami xture
result
sei t
herbyadsor ptionorpar t
iti
onf orthecol
umnmat erial
.Theusualtype
ofc hr
omat ographyinvolvesmovementofl i
qui
dphaseoverst at i
onaryphase
carryi
ngwi thitasol ut
ewhi c
hhasvar yingdegreeofaf f
ini
tyf orstati
onary
phase.Thusdependi ngupont hephases,chromatographyisclassif
iedonthe
foll
owingbasis:

1
. Par
tit
ionc
hromat
ogr
aphy:
-

Tahi
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 Thisi
nvol
vesl
iqui
dorgasasmobi
lephaseandanot
herl
iqui
dorsol
idasa
stati
onar
yphase.

Theoper
ati
oni
ncl
udes:

i
)Par
tit
ionc
olumnc
hromat
ogr
aphy

i
i)Paperc
hromat
ogr
aphy

i
ii
)Thi
nLayerc
hromat
ogr
aphy

i
v)Gas-
li
qui
dchr
omat
ogr
aphy

v)
Highper
for
manc
eli
qui
dchr
omat
ogr
aphy

2. Adsor
pti
onc
hromat
ogr
aphy:
-

Thisi
nvol
vesl
iquidorgasasmobil
ephaseandadsor
bentsol
idas
st
ati
onar
yphase.Thet ypesundert
hisc
ategoryi
ncl
ude:

i
)Adsorpti
onc
olumn
c
hromatography
. i
i)Thi
n
l
ayer
c
hromat
ogr
aphy
. i
ii
)Gas
-
sol
idc
hromat
ogr
aphy

Di
str
ibut
ionofanal
ytesbet
weenphases
Thedi
stri
buti
onofanalytesbetweenphasesc
anof t
enbedesc
ribedqui
tesi
mpl
y.
Ananal
yteisinequi
li
bri
umbet weenthetwophases;

Partiti
onc oeffi
cient
:-
I
ft hemobi lephasef l
owisstoppedatanytime,thesol
uteassumesanequi
li
bri
um
distr
ibutionbetweent hetwophases.Theconcentr
ati
onineachphasei
sgivenby
thet hermodynami cpart
iti
oncoeffi
cient
;
K=  CS/CM 

WhereCS andCM  aretheconcentr
ati
onofsol
uteinthest at
ionar
yandmobil
e
phases,r
espec
tivel
y.WhenK=1,thensol
utei
sequal
lydist
ributedbetweent
he
twophase.

Forasymmetri
calpeak,whenthepeakmaxi
mumappearsatthecol
umnexi
st,hal
f
ofthesol
utehaselutedintheret
enti
onvol
ume,VR ,andhalfr
emain

Tahi
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0
dist
ribut
edbetweent hevolumeoft
hemobi
lephase,
VM , andt
hevol
umeoft
he
stati
onaryphase,VS, thus,
VRCM =VMCM  +VSCS

Rear
rangementandc
ombi
nat
ionwi
thupperequat
ion,
VR =VM  +KVS

I
tr el
atestheretentionvolumeofsolut etothecolumndeadvol umeandt he
productofthepar t
itioncoeff
ici
entandt hevolumeofst ati
onaryphase.Thi
s
equati
onc or
rectforl i
qui
dpar ti
tionc
olumn,butf oradsorpti
onc olumn,
1
VS shouldbereplacedbyAS, t hesurfacear
eaoft headsorbent.

Part
iti
onr at
io:
-
Theparti
tionrato K’,
i i
st hemosti mport
antqual
ityi
ncol
umnc hromat ography.
Foragivensetofoperati
ngpar ameterK’
,   isameasureofthet imespenti n
thestati
onaryphaserel
ati
vet ot heti
mespentinthemobi
lephase.Itisdefined
astheratioofmolesofsolutesinthestati
onar
yphasetothemol esint he
mobil
ephase: 

k'
 =CSVS/CMVM  =K(VS/VM)

t
hevol
umet
ricr
ati
o,VM/VS,i
sof
tendenot  K’=
edbysymbolβ.Thus,  K/β.

Statedanot
herway,thepar
tit
ionrati
oistheaddit
ionalt
imeasolutebandtakes
toelute,ascompar
edwithanunretainsol
utedivi
dedbyt heel
uti
ontimeofan
unretai
nband.

k'
= t
 R -
 tM / t
M   =VR  -
  VM /VM

therelat
ionstatesexpl
ici
tyhowmanydeadvol
ume(ort
M )arer
equi
redt
o
attai
nVR ( ortR).

Pl
ateTheor
y
Theplat
emodelsupposest hatt hechromatographiccolumniscontai
nsal arge
numberofseparatel
ayers,called theoretic
alplates.Separ
ateequil
ibrati
onsof
thesamplebetweenthestationaryandmobi lephaseoc curi
nthese"plates".The
anal
ytemovesdownt hecolumnbyt ransferofequili
bratedmobil
ephasef rom
oneplat
et othenext
.

Tahi
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(U.
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Itisimportanttor emembert hatt heplatesdonotreallyexist;theyarea
figmentoft heimagi nationthathelpsusunderstandthepr ocessesatwor ki
nthe
column.Theyalsoser veasawayofmeasur i
ngcolumneffic
iency,eitherby
statingthenumberoft heor
et i
calplat
esi nacol
umn, N ( themor eplatesthe
better),orbyst atingt hepl
at eheight;the Hei
ghtEquivalentt oaTheoreti
cal
Plate ( t
hesmal lert hebetter).

I
fthel
engt
hoft
hec
ol s L,t
umni hent
heHETPi
s

 L/N
HETP=

Theseparationeffi
ciencyofc hromatographiccol
umni ncreaseswithi
ncreases
numberoft heoret
icalplates.Thusthenumberoft heoreticalpl
ates‘
N’isusedas
measureofc ol
umnef f
ici
ency.Theoreti
calplatescanbeeasi l
ymeasuredfromt he
chr
omat ogr
am.t henumberoft heoreti
calplates‘N’isgivenby

2
N=1
6(t
/w)

Wher
e‘t’i
sthedi
stantfr
ominj
ect
iont
opeakmaxi
mum(r
etent
iont
ime)and‘
W’
i
sthepeakwi
dthi
nunitsoft
ime.

Now,

2
HETP=L/N=L/1
6(w/t
)

Tahi
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Wher
e“L” isthelengthofchr
omat ogr
aphicc
olumn,i ncent
imet
er.Thus,HETP
4
andNarethepref
erredmeasuresofcolumneffi
cienc
y.

Separ
ati
onTec
hni
ques
Chr
omat
ogr
aphi
csepar
ati
onc
anbec
arr
youtbyt
hef
oll
owi
ngt
echni
ques:

El
uti
onanal
ysi
s:
I
tisver ycommonmet hodusedinthecol
umnc hromatography.Inthi
smethoda
smallvolumeofmi xtur etobeisaddedont het opofc ol
umnandmobi l
ephaseis
all
owedt oflowthr oughc ol
umn.Asmobilephasedownt hec olumn,t
hemixture
int
roducedont hec olumnget ssepar
atedintoz onesast hecomponentoft he
mixtureareadsorbedt othecol
umnmat erialt
odi ffer
entext end.

Onfurtherpassageofmobil
ephase,eachc
omponentofmi xtur
e  iselut
edout
asseparatedcomponent.Apl
otofgraphofamountsepar
at edoutversusvol
ume
ofelut
edorf racti
ons.
 

Fr
ont
alanal
ysi
s:

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I
nthismethod,t
hesoluti
onofthesampl
emixt
ureisaddedconti
nuousl
yont
he
c
olumn.Nomobil
ephseisusedf
orthedevel
opmentofcolumn.

Thismet hodcanbeunder stoodbybyt heconti

nuousaddi tionoft hesampl e
mixtur eofAandBont ot hec olumnwhi chisfi
ll
edwi thaknownamountoft he
adsor bent.Afterc ertai
nt i
met hemi xtureisadsorbedc ompl etel
yandt hecolumn
saturat ed.Then,onc ont ainingt headdi ti
onoft hemi xt ure,t hel
eesst r
ongly
adsor bedc omponentAl eavest hec olumni nthepur ef orm.Theni ti selutedasa
mixtur ewi thcomponentBwhi chget sadsobedmor est ronglyandc onsequently
movesmor eslowly.TheamountofAi nthemobi l
ephasec anbec alculatedfrom
ther etent i
onvol umeVR, whi l
eot herc omponentc anbedet er mi
nef romt he
volumec oncentrationrat io.Compl etesepar ati
onoft hec omponentc annotbe
achived,whenmor ethant woc omponentar epresenti nthesampl et obe
separ ated.

   Di spl
acementanalysis:
I
nt hi
smet hod,asmallquantityofsamplesoluti
onfirstintroduc
edatt hetopof
thecolumn.Thec omponentoft hemixturearethensepar atedbyrunninga
sol
utionofasubst ancewhichismorest r
onglyadsorbedt hananyoft he
componentsoft hemixture.Thesubstancethusrunist ermedas displasi
ng
agent.

Thisc anbeundr est

oodbyi nvol vi
ngt heel uti
onordevel opmentbyanel uantC
whichi shavi ngagr eateraf f
inityf orthest ati
onar yphaset hant hesampl e
component sAandB(A> B) .thesampl emixturec ontaini
ngAandBi sf i
rst
introduc edatt hetopoft hec olumnwher eitadher et ot hestationaryphase.
Elutiont akepl acewhent hedi splacingeluantc ontai
ningCi spassedt hrought he
columndi splac
ingt hecomponent sont hestationaryphase.Dur ingt hepr ocess
thec omponentunder goessepar at i
onbec auseoft hedi fferenceint hepar ti
tion
oradsor ptionpr operti
es.thismet hoddoesnotgener all
ypr oducedc ompl et
ely
separ atedc omponent .

Tahi
raI
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ienc
es,
GIDA,Gor
akhpur
(U.
P)Page1
4
 Col
umnChr
omat
ogr
aphy

I
nchemi
stry,columnchromatogr
aphyisatechniquewhi
chisusedtosepar
atea
si
ngl
echemicalcompoundfromamixturedi
ssolvedinafl
uid.

Columnc hromatographysepar atessubst ancesbasedondif

ferentialadsorption
ofc ompoundst otheadsor bentast hec ompoundmovest hrought hecolumnat
dif
f er
entr at
eswhi chall
owst hemt ogetsepar atedi
nfracti
ons.Thist echni
que
canbeusedonasmal lscaleaswel laslargesc al
etopuri
fymat erial
st hatcanbe
usedinf utureexperi
ment s.Thismet hodi satypeofadsorpti
onc hromat ography
technique.

#Pr
inc
ipl
e:-
Whent hemobi l
ephasealongwitht hemixturet hatneedstobesepar atedis
i
ntroducedfromt hetopoft hecolumn,themovementoft heindi
vidual
componentsoft hemixturei
satdi ffer
entr ates.Thecomponent swithlower
adsorpti
onandaf fi
nitytothestat i
onaryphaset r
avelfasterwhenc omparedt
o
thegreateradsorpti
onandaf fi
nit
ywi ththest ationar
yphase.Thec omponents
thatmovesf asterareremovedfirstwhereast hec omponentsthatmovesl owl
y
areelutedoutlast.

Theadsorpt
ionofsol
utemolec
ulest
ot hecolumnoccur
sinarever
sibl
emanner
.
Therateofthemovementofthecomponentsisexpr
essedas:

Rf
=thedi
stanc
etr
avel
ledbysol
ute/t
hedi
stanc
etr
avel
ledbyt
hesol
vent

Rfi
sther
etar
dat
ionf
act
or.

Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
GIDA,Gor
akhpur
(U.
P)Page1
5
 Fi
g:c
olumnc
hromat
ogr
aphydi
agr
am

#Pr
ocedur
e:-
Befor
estart
ingwitht
hecolumnc
hromat
ogr
aphyexper
iment
,letusunder
stand
thedi
ffer
entphasesi
nvol
ved.

Mobil
ePhase-Thi
sphasei
smadeupofsol
vent
sandi
tper
for
mst
hef
oll
owi
ng
func
tions:

1
.Itac
tsasasol
vent
-sampl
emi
xtur
ethatc
anbei
ntr
oduc
edi
nthec
olumn.

2.I
tac
tasadevel
opingagenti
.e,hel
psi
nthesepar
ati
onofc
omponent
sin
t
hesampl
etoformbands.

3.I
tac
tsasaneluti
ngagenti
.e,thec
omponentsthatar
esepar
ateddur
ing
t
heexper
imentarer
emovedf r
omt hec
olumn.

4.Someexampl
esofsol
vent
susedasmobi
lephasesbasedont
hei
rpol
ari
ty
areEt
hanol
,Acet
one,Water
,Ac
eti
caci
d,Pyr
idi
ne,etc.

Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
GIDA,Gor
akhpur
(U.
P)Page1
6
St
ati
onaryPhase-I
tisasol
idmat er
ialwhi
chshoul
dhavegoodadsor
pti
on
pr
opert
iesandmeetthecondi
tionsgivenbel
ow:

1
. Shapeandsizeofpart
icl
es:par
tic
lesshoul
dhaveauni
for
mshapeandsi
ze
i
nt herangeof60-200micronindiamet
er.

2.Stabi
li
tyandi nertnessofpart
icl
es:hi
ghmechani
calstabi
li
tyandchemi
cal
ly
i
nert.Also,nor eacti
onwithaci
dsorbasesoranyot hersol
vent
swasused
duri
ngt heexper i
ment .

3.I
tshoul
dbec
olour
less,i
nexpensi
veandr
eadi
lyavai
labl
e.

4.Shoul
dal
lowf
reef
lowofmobi
lephase.

5.I
tshoul
dbesui
tabl
efort
hesepar
ati
onofmi
xtur
esofvar
iousc
ompounds.

#Col
umnc
hromat
ogr
aphyExper
iment
:
>St ati
onaryphaseismadewetwi t
ht hehelpofsolventast heupperl evelof
mobilephaseandt hest ati
onaryphaseshoul dmatch.Themobi lephaseorel uent
iseithersol
ventorami xtureofsolvents.I
nt hef
irststepthec ompoundmi xture
thatneedst obesepar ated,isaddedfromt hetopoft hecolumnwi thout
dist
ur bi
ngthet oplevel.Thetapist ur
nedonandt headsor ptionprocessont he
surfaceofsili
cabegins.

>Withoutdist
ur bi
ngthestati
onaryphasesol
ventmixt
ureisaddedslowl
yby
touchingthesidesoftheglasscol
umn.Thesolventi
saddedthroughoutt
he
experimentaspert herequi
rement.

>Thetapisturnedont
oi ni
tiat
et hemovementofc ompoundsint hemixt
ur e.The
movementisbasedonthepolari
tyofmoleculesi
nt hesample.Thenon-polar
component
smoveatagr eaterspeedwhenc omparedtothepol arcomponent s.

>Forexampl
e,acompoundmixtureconsi
stsofthr
eedif
ferentcompoundsvizr
ed,
blue,gr
eenthenthei
rorderbasedonpolari
tywil
lbeasfoll
owsblue>
red>gr
een.

>Asthe polar
ity oft
hegreencompoundisl
ess,i
twillmovefi
rst.Whenit
arri
vesattheendofthecol
umnitiscol
lec
tedi
nac leantestt
ube.Afterthi
s,t
he

Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
GIDA,Gor
akhpur
(U.
P)Page1
7
r
edcompoundiscol
lec
t edandatlastbl
uec
ompoundi
scol
lec
ted.Al
lthesear
e
c
oll
ect
edinsepar
atet esttubes
.

#Col
umnc
hromat
ogr
aphyappl
icat
ions:

 Col
umnChr
omat
ogr
aphyi
susedt
oisol
ateac
tivei
ngr
edi
ent
s.

 I
tisver
yhel
pfuli
nsepar
ati
ngc
ompoundmi
xtur
es.

 I
tisusedt
odet
ermi
nedr
ugest
imat
ionf
romdr
ugf
ormul
ati
ons.

 I
tisusedt
oremovei
mpur
iti
es.

 Usedt
oisol
atemet
abol
itesf
rombi
ologi
calf
lui
d.

#Typesofc
olumnChr
omat
ogr
aphy:

 Adsorpti
onc
olumnc
hromat
ogr
aphy–Adsor
pti
onc
hromat
ogr
aphyi
s
atec
hni
queofseparati
on,inwhi
chthecomponent
soft
hemi
xtur
e
ar
eadsorbedonthesurfac
eoftheadsorbent
.
 Parti
tionc
olumnc
hromat
ogr
aphy–Thest
ati
onar
yphase,aswel
las
mobi
lephase,ar
eli
qui
din par
tit
ionc
hromat
ogr
aphy.
 Gelcol
umnc
hromat
ogr
aphy–I
nthi
smet
hodofc
hromat
ogr
aphy,t
he
separat
iontakesplac
ethroughacol
umnpackedwi
thgel.The
stati
onaryphaseisasol
venthel
dinthegapofasolvent
.
I
onexc
hangec
olumnc
hromat
ogr
aphy–Ac
hromat
ogr
aphyt
echni
que
i
nwhi
cht
hest
ati
onar
yphasei
sal
waysi
onexc
hanger
esi
n.

PaperChr
omat
ogr
aphy

Chromatographytechni
quet
hatusespapers
heetsorstr
ipsastheadsorbent
bei
ngt hestati
onaryphaset
hroughwhic
has ol
uti
onismadetopassiscall
ed
paperchromatography.
I
tisaninexpensi
vemethodofseparati
ngdissol
vedc
hemi
calsubst
anc
esbyt
hei
r
dif
ferentmigrati
onr
atesacros
st hesheet
sofpaper.
I
tisapower
fulanal
yti
calt
oolt
hatusesver
ysmal
lquant
iti
esofmat
eri
al
Paperc
hromat
ogr
aphywasdi
scover
edbySyngeandMar
tini
ntheyear1
943.

Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
GIDA,Gor
akhpur
(U.
P)Page1
8
#Pr
inc
ipl
e:

Thepr
inci
pleinvol
vedc
anbepar
tit
ionc
hromat
ogr
aphyoradsor
pti
on
chr
omatography.

Parti
tionc
hromatographybec
auset hesubst
ancesarepart
iti
onedordistri
but
ed
betweenli
qui
dphases.Thetwophasesar ewaterhel
dinporesofthefilt
erpaper
andtheotherphaseisamobilephasewhichpassest
hroughthepaper

Whent hemobil
ephasemoves,the separ
ationoft hemi
xt ur
e t akesplac
e.The
compoundsinthemixtur
eseparatet
hemselvesbasedont hedifferencesinthei
r
affi
nit
yt owar
dsstati
onaryandmobil
ephasesolventsundert hec api
ll
aryacti
on
ofporesinthepaper.

Adsorpti
onchr
omatographybetweensoli
dandli
quidphases,wher
einthesol
id
surfaceoft
hepaperisthestat
ionaryphaseandtheli
quidphaseisthemobi
le
phase.

Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
GIDA,Gor
akhpur
(U.
P)Page1
9
 f
ig:paperc
hromat
ogr
aphydi
agr
am

#Paperc
hromat
ogr
aphypr
ocedur
e:

1
. Selec ti
ngasui tablet ypeofdevel opment :
 I tisdec idedbasedont he
compl exityoft hesolvent ,paper ,mi xtur e,et c.Usual lyasc endingt ypeor
radialpaperc hromat ogr aphyi susedast heyar eeasyt oper form.Al so,itis
easyt ohandl e,thec hromat ogr amobt ainedi sf asterandt hepr oc essi sless
time- consumi ng.
2.Selec ti
ngasui tablef i
lterpaper :Sel ec ti
onoff i
lterpaperi sdonebasedont he
sizeoft hepor esandt hesampl equal i
ty.
3.Prepar et hesampl e: Sampl epr epar ationi ncludest hedi ssolutionoft he
sampl einasui tablesolvent( iner twi tht hesampl eunderanal ysis)usedi n
maki ngt hemobi l
ephase.
4.Spott hesampl eont hepaper : Sampl esshoul dbespot tedatapr oper
posit i
onont hepaperbyusi ngac api ll
ar yt ube.
5.Chromat ogr am devel opment : Chr omat ogr amdevel opmenti sspot t edby
immer singt hepaperi nt hemobi lephase.Duet ot hec apil
lar yactionofpaper ,
themobi lephasemovesovert hesampl eont hepaper .
6.Paperdr yingandc ompounddet ec t ion:Onc et hec hromat ogr ami sdevel oped,
thepaperi sdr iedusinganai rdr ier .Also,det ect i
ng sol ut ion c anbespr ayed
ont hec hr omat ogramdevel opedpaperanddr i
edt oi dentifyt hesampl e
chromat ogramspot s.

#Paperc
hromat
ogr
aphyappl
icat
ions:

Therearevari
ous appl
icat
ionsofpaperchr
omatography.Someoft
heusesof
PaperChromatogr
aphyi
ndifferentf
iel
dsaredi
scussedbelow:

Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
GIDA,Gor
akhpur
(U.
P)Page20
  Tost
udyt
hepr
ocessoff
erment
ati
onandr
ipeni
ng.

 Toc
hec
kthepur
ityofphar
mac
eut
ical
s.

 Toi
nspec
tcosmet
ics.

 Todet
ectt
headul
ter
ant
s.

 Todet
ectt
hec
ont
ami
nant
sindr
inksandf
oods.

 Toexami
net
her
eac
tionmi
xtur
esi
nbi
ochemi
call
abor
ator
ies.

 Todet
ermi
nedopesanddr
ugsi
nhumansandani s.
mal

#TypesofPaperChr
omat
ogr
aphy:

1
. Ascendi ngPaperChr omat ography–Thet echniquesgoeswi thi tsnameast he
solventmovesi nanupwar ddi rection.
2.Desc endingPaperChr omat ogr aphy–Themovementoft hef lowofsol ventdue
togr avit at
ionalpul landc api
llaryac tioni sdownwar ds,henc ethename
descendi ngpaperc hr omat ography.
3.Ascendi ng–Desc endi ngPaperChr omat ography–I nt hisver sionofpaper
chromat ography,movementofsol ventoc cursint wodi rectionsaftera
particularpoi nt.I
nit i
ally,thesol ventt ravelsupwar dsont hepaperwhi chis
foldedoverar odandaf terc rossingt her oditc ontinueswi t hi
tstr avelinthe
downwar ddirecti
on.
4.RadialorCi r
c ul
arPaperChr omat ogr aphy–Thesampl eisdeposi tedatt he
centr eoft hec ircularfilterpaper .Onc et hespoti sdr ied,thef il
terpaperi s
ti
edhor izont al
lyonaPet ridishwhi chc ont ai
nst hesol vent.
5.TwoDi mensi onalPaperChr omat ogr aphy–Subst anceswhi chhavet hesame
rf val uesc anber esolvedwi tht hehel poft wo- di
mensi onalpaper
chromat ography.

Gas–l
iqui
dChr
omat
ogr
aphy

Gas-
Liqui
dChr
omat
ogr
aphy(
GLC)i
sa separ
ati
ont
echni
que i
nwhi
chgas
(
usual
lyi
ner
tgas,suc
hashel
iumornonr
eac
tivegas,suc
hasni
trogen)
,isused

Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
GIDA,Gor
akhpur
(U.
P)Page21
asamobi
lephase,andl
iqui
dasast
ati
onar
yphase.
Basi
sofsepar
ati
oni
sdi
ffer
enc
einpar
tit
ionc
oef
fic
ientofvol
ati
li
zedc
ompounds
bet
weenl
iqui
dandgasphaseswhent
hedesi
redc
ompoundi
scar
riedt
hrought
he
c
olumnbyac
arr
iergas.

Typi
calappl
icat
ionofGLCi
ncl
udest
est
ingpur
ityofasubst
anc
e,pr
epar
enew
c
ompoundsf
rommi
xtur
e(pr
epar
ati
vec
hromat
ogr
aphy)
,ident
ifyi
ngand
separ
ati
ngdi
ffer
entc
omponent
sofami
xtur
e.
I
tisal
soknownasVapor
-PhaseChr
omat
ogr
aphy(
VPC)
,orGas-
Liqui
dPar
tit
ion
Chr
omat
ogr
aphy(
GLPC)
.

#Pr
inc
ipl
e:

GLCi
sbaseduponpar
tit
ioni
ngofc
ompoundsbet
weenst
ati
onar
yli
qui
dand

Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
GIDA,Gor
akhpur
(U.
P)Page22
 mobi
legasphase.Duet
oit
shi
ghsensi
tivi
ty,r
epr
oduc
ibi
li
ty,andspeedof
r
esol
uti
on,i
tiswi
del
yusedf
orsever
alqual
itat
iveandquant
itat
iveanal
yses.
Thegaseousc
ompoundsbei
nganal
yzedi
nter
actwi
tht
hewal
lsofc
olumn,whi
chi
s
c
oat
edwi
thdi
ffer
entki
ndsofst
ati
onar
yphases.
Thi
scauseseac
hcompoundt
oel
uteatdi
ffer
entt
ime(
ret
ent
iont
ime)
,andt
hen
r
etent
iont
imesar
ecompar
ed.Thi
smakesGLCanal
yti
cal
lyver
yusef
ul.
Asc
ompoundl
eavest
hec
olumn,t
heypasst
hroughadet
ect
or,whi
chi
sli
nkedt
o
c
har
trec
ordervi
aampl
ifi
er.Char
trec
orderr
ecor hepeaks.
dst

#Appl
icat
ions:

Gasl
iqui
dchr
omat
ogr
aphyhasaver
ywi
def
iel
dofappl
icat
ioni
nthesepar
ati
on
andanal
ysi
sofmul
tic
omponentmi
xtur
essuc
hasessent
ialoi
ls,hydr
ocar
bons
andsol
vent
s.I
tisoneoft
hepr
imar
yanal
yti
calt
echni
ques,whi
chwasusedi
n
f
orensi
clabor
ator
y.
 GLCi
scombi
nedwi
thmassspec
trosc
opy(
GC/MS)f
ordr
ugdet
ect
ion,f
ire
i
nvest i
gat i
on,environmentalanalysi
s,expl
osi vesinvesti
gation,and
i
dent i
fi
c ationofunknownsampl es.
 GC/MSi sal sousedi nai
rportsecuritytodet ectunwant edsubstances.Non
deri
vat i
z edsugarsandsugaral c
ohol saresuc cessful
lyanalyzedbyGC/MSusi ng
atmospher icpressurechemicali
onizati
on( APCI)innegat i
veionmode.
 GCiswi delyusedbyf or
ensicsci
ent i
stsforananal ysisofbodyf lui
dsf orthe
presenceofi ll
egalsubstances,testi
ngoff iberandbl oodfromac r
imesc ene,
andt odet ectresiduefromexplosives.

#Pr
ocedur
e:
Aflowdi
agramrepr
esenti
ngthest
epsi
nvol
vedi
nthepr
ocessofgas-
li
qui
d
chr
omatogr
aphyisshownbel
ow:

 
Sel
ect
ionofc
olumns,st
ati
onar
yphase,i
nlett
ypes,

Car
riergas,andt
hei
rfl
owr
ates
Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
GIDA,Gor
akhpur
(U.
P)Page23
 Addi
tionoft
hesampl
est
obei
njec
tedt
othesyr
inge

I
njec
tionoft
hesampl
eint
othei
njec
torpor
t

Dat
areduc
tionandanal
ysi
s

Thi
nLayerChr
omat
ogr
aphy

Thi
nLayerChromatographyi satec
hniqueusedt oisol
atenon- volat
ilemixt
ures.
Theexperi
mentisconductedonasheetofal umi
niumfoil
,plastic,orglasswhic
h
i
scoatedwithathinlayerofadsorbentmat eri
al.Thematerialusuall
yused
i
s alumini
umoxide,cell
ulose,orsi
li
cagel.

Oncomplet
ionofthesepar
ati
on,eac
hcomponentappearsasspot
ssepar
ated
ver
tic
all
y.Eachspothasaret
enti
onfac
tor(Rf)expr
essedas:

Rf =di
st.t
ravel
ledbysampl
e/di
st.t
ravel
ledbysol
vent
Thef actorsaffectingr
etardationfactoraret hesolventsyst
em,amountof
mater i
alspotted,adsorbentandt emperature.TLCisoneoft hefast
est,l
eas
t
expensive,si
mpl estandeasiestchromatographyt ec
hnique.

Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
GIDA,Gor
akhpur
(U.
P)Page24
#Pr
inc
ipl
e:

Li
keotherchromat
ographictec
hni
ques,t
hin-
layerc
hromat
ogr
aphy(
TLC)depends
ontheseparati
onpri
nci
ple.
Theseparat
ionrel
iesontherel
ati
veaffini
tyofc
ompoundst
owar dsbot
hthe
phases.Thecompoundsi
nt hemobi
lephasemoveovert
hesurfac
eoft he
stati
onaryphase.
Themovementoc c
ur sinsuchawayt hatthecompoundswhic
hhaveahi gher
affi
nit
yt othestationaryphasemoveslowlywhi
letheothercompoundstravel
fast,the separ
ationoft hemixt
ure isattai
ned.
Oncompleti
onoft hes
eparationprocess,t
heindivi
dualcomponentsfromthe
mixtureappearasspot
satr especti
velevel
sont heplates.Thei
rchar
ac t
erand
natureareident
ifi
edbysui
tabl edet
ectiontec
hniques.

Fi
g:t
hinl
ayerc
hromat
ogr
aphy

#Pr
ocedur
e:
Befor
estartingwi
tht heThi
nLayerChr
omatogr
aphyExperi
ment
,letus
under
standthediff
erentcomponent
srequi
redtoconduc
ttheprocedur
eal
ong
withthephasesi
nvolved.

Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
GIDA,Gor
akhpur
(U.
P)Page25
 1
. ThinLayerChr omat ogr aphyPl ates–r eady-madepl atesar eusedwhic har e
chemicallyiner tandst able.Thest ationar yphaseisappliedoni tssurf ac e
i
nt hef ormofat hinlayer .Thest at i
onar yphaseont hepl atehasaf ine
parti
clesi zeandal sohasauni formt hickness.
2.ThinLayerChr omat ogr aphyChamber–Chamberi susedt odevel opplat es.
I
tisresponsi bletokeepast eadyenvi ronmenti nsi
dewhi chwi l
lhelpi
n
devel
opi ngspot s.Also,i tprevent st hesol ventevapor ationandkeepst he
enti
repr oc essdust -free.
3.ThinLayerChr omat ogr aphyMobi l
ephase–Mobi l
ephasei stheonethat
movesandc onsist sofasol ventmi xtureorasol vent.Thisphaseshoul dbe
parti
culat e-free.Thehi ghert hequal ityofpur i
tyt hedevel opmentofspot s
i
sbet ter.
4.ThinLayerChr omat ogr aphyFilterPaper–I thast obepl acedinsidet
he
chamber .Itismoi st enedi nt hemobi l
ephase.

#Thi
nlayerc
hromat
ogr
aphyExper
iment
:
Thest
ati
onar
yphaset
hati
sappl
iedt
othepl
atei
smadet
odr
yandst
abi
li
ze.

 Toappl
ysampl
espot
s,t
hinmar
ksar
emadeatt
hebot
tomoft
hepl
ate
wi
tht
hehel
pofapenc
il.

 Appl
ysampl
esol
uti
onst
othemar
kedspot
s.

 Pourt
hemobi
lephasei
ntot
heTLCc
hamberandt
omai
ntai
nequalhumi
dit
y,
pl
aceamoi
stenedf
ilt
erpaperi
nthemobi
lephase.

 Pl
acet
hepl
atei
ntheTLCc
hamberandc
losei
twi
thal
id.I
tiskepti
nsuc
ha
wayt
hatt
hesampl
efac
est
hemobi
lephase.

 I
mmer
set
hepl
atef
ordevel
opment
.Remembert
okeept
hesampl
espot
s
wel
labovet
hel
eveloft
hemobi
lephas
e.Donoti
mmer
sei
tint
hesol
vent
.

 Wai
tti
llt
hedevel
opmentofspot
s.Onc
ethespot
sar
edevel
oped,t
akeout
t
heplatesanddr
ythem.Thesampl
espot
scanbeobser
vedunderaUV
l
ightc
hamber.

#Thi
nlayerc
hromat
ogr
aphyappl
icat
ions:

 Thequal
itat
ivet
est
ingofVar
iousmedi
cinessuc
hassedat
ives,l
ocal
anaestheti
cs,ant
iconvul
santt
ranqui
li
ser
s,anal
gesi
cs,ant
ihi
stami
nes,
steroi
ds,hypnoti
csisdonebyTLC.

 TLCi
sext
remel
yusef
uli
nBi
ochemi
calanal
ysi
ssuc
hassepar
ati
onor
i
sol
ati
onofbi
ochemi
calmet
abol
itesf
romi
tsbl
oodpl
asma,ur
ine,body

Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
GIDA,Gor
akhpur
(U.
P)Page26
 f
lui
ds,ser
um,et
c.

 Thi
nlayerc
hromat
ogr
aphyc
anbeusedt
oident
ifynat
uralpr
oduc
tsl
ike
ess
ent
ialoi
lsorvol
ati
leoi
l,f
ixedoi
l,gl
ycosi
des,waxes,al
kal
oids,et
c.

 I
tiswi
del
yusedi
nsepar
ati
ngmul
tic
omponentphar
mac
eut
icalf
ormul
ati
ons.

 I
tisusedf
ort
hepur
ifi
cat
ionofsampl
esanddi
rec
tcompar
isoni
sdone
bet
weent
hesampl
eandt
heaut
hent
icsampl
e.

 I
tisusedi
nthef
oodi
ndust
ry,t
osepar
ateandi
dent
ify
c
olour
s, sweet
eni
ngagent
,andpr
eser
vat
ives

 I
tisusedi
nthec
osmet
ici
ndust
ry.

 I
tisusedt
ost
udyi
far
eac
tioni
scompl
ete.

#Di
sadvant
agesofThi
nlayerc
hromat
ogr
aphy:

1
. ThinLayerChr omatographyplatesdonothavel ongerst ationar
yphase.
2.Whenc omparedt ootherchromatographi ctechniquesthel engthof
separat i
onisli
mited.
3.Ther esultsgeneratedfromTLCar edi ff
icul
tt or eproduce.
4.SinceTLCoper atesasanopensyst em,somef actorssuc hashumi di
tyand
temper aturecanbec anaffectthef i
nalout comeoft hec hromatogram.
5.Thedet ectionli
mitishighandther ef
or eifyouwantal owerdet ect
ionl
imi
t,
youc annotuseTLC.
6.I
ti sonlyaqual i
tati
veanalysistechniqueandnotquant i
tative.

Hi
ghPer
for
manc
eThi
nLayerChr
omat
ogr
aphy
*Sophist
icat
edf ormoft
hinlayerc
hromat
ography.I
tinvol
vest
hesame
theoreti
calpri
ncipl
eofthi
nlayerchr
omatogr
aphy.

*I
tisal
soknownaspl
anarc
hromat
ogr
aphyorFl
at-
bedc
hromat
ogr
aphy.

#Pr
inc
ipl
e:
>HPTLChavesimi
larapproac
handempl
oyt
hesamephysi
calpr
oper
tiesofTLC
(
adsorpt
ionchr
omat ogr
aphy).

>Themobi
lephasesol
ventf
lowst
hroughbec
auseofc
api
ll
aryac
tion.The

Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
GIDA,Gor
akhpur
(U.
P)Page27
 componentsmoveaccordi
ngt othei
raffi
niti
est owardst headsorbent.The
componentwit
hmor eaffi
nitytowardsthest at
ionaryphaset ravelssl
ower.The
componentswit
hlesseraffi
nit
yt owar
dst hestationaryphaset ravel
sfaster
.
Thusthecomponentsareseparatedonac hromatogr aphicpl
ate.

#Pr
ocedur
e:
Sel
ect
ionofc
hromat
ogr
aphyl
ayer
Sample
prepar
ati
on

Pr
e-washi
ng

Appl
icat
ionofsampl
e Pr
e-c
ondi
tioni
ng

Chromat
ography
devel
opment

Sc
anni
nganddoc
ument
ati
on

Det
ect
ionofspot
Appl
icat
ionsofHPTLC:

#Appl
icat
ionsofHPTLC:

 Pharmaceuti
cali
ndust
ry:qual
ityc
ont
rol
,cont
entuni
for
mit
y,
uni
for
mit
ytest
,ident
ity/pur
ityc
hec
k.

 FoodAnal
ysi
s:qual
ityc
ont
rol
,addi
tives,pest
ici
des,st
abi
li
tyt
est
ing,
anal
ysi
sofsub-
mic
ronl
evel
sofaf
lot
oxi
ns,
etc
.

 Cl
ini
calAppl
icat
ions:met
abol
ismst
udi
es,dr
ugsc
reeni
ng,st
abi
li
ty

Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
GIDA,Gor
akhpur
(U.
P)Page28
 t
est
ing,
etc
.For
ensi
c:poi
soni
ngi
nvest
igat
ion.

Conc
lusi
on

Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
GIDA,Gor
akhpur
(U.
P)Page29
 Ref
erenc
e

Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
GIDA,Gor
akhpur
(U.
P)Page30
 1
. Set
hiPD,HPTLCHi ghPerfor
manc eLiqui
dChr
omat
ogr
aphy,Fi
rst
edi
tion,CBSPubl
isherandDist
ributers.

2.Rei
ch,E.andSchhi
blia.
(2007)Hi
ghPerformanc
eLiquid
Chr
omat ogr
aphyforanalysi
sofmedi
cinalpl
ant,Thi
eme.

3.ArupU,EkmanS,Lindbl
omL,MattssonJ
E.Hi
ghPer
for
manc
eLi
qui
d
Chromat
ography(HPTLC),1
993.

4.www.
phar
mai
nfo.
com

5.Dr.HarishChandraAndola,“Hi
ghPerformanceLiqui
d
Chromat ogr
aphy(HPTLC):AModer nAnalyt
icalt
oolforBi
ologi
cal
Analysi
s”,NatureofScience,
2010

6.PatelR.
B.andPatelM.
R.andPatelB.
G( 2011
)Experi
ment
alaspec
ts
andimpl
ementati
onofHPTLC,NewYor k,Spri
nger.

7.Shri
vast
avaM.
M.(
201
1
),anover
viewofHPTLC:AModer
nAnal
yti
cal
Techni
quewit
hExcel
lentPotenti
alforAutomation,Opt
imi
zat
ion,
Hyphenat
ionandMul
t i
dimensi
onalAppl
icat
ions.

8.    Caz esJ,sc

ot tRPW,chr
omatographyt
heor
y,NewYor
k.basal
:
mar
celdekker
,inc.pageno–261-
285.

9. ChatwalGR,anandSK,I nt
rumentalmethodsofchemi
calanal
ysi
s,
edi
tedbyM.arora,Aseemanand,Mumbai:
Himal
ayapubli
shi
nghouse,
pageno-2.
556-2.585

1
0.  
 und.edu/dept
/c hromatogr
aphy/doc
ument
s/Chr
omat
ogr
aphy.
pdf
..
Ac
cessedon25/1 2/2010

1
1
.  
 teachi
ng.
shu.
ac.uk/hwb/c
hemi
str
y/t
utor
ial
s/c
hrom/c
hrom1
.ht
m.
Ac
cessedon25/12/2010

1
2.    J effery,bassett,ment ham,denney,vogel’
st ext
bookof
quant
itati
vec hemicalanalysi
s,fi
fthedi
tion,London:theschoolof
chemi
stry.Pageno–21 6-221.

1
3.    Kast ureAV,mahadi
kKR,wadodkarSG,moreAH,
Pharmac
eutic
alamal
ysi
svol-2i
ntr
umentalmet
hod,ni
ral
ipr
akashan.
pageno-6-
9,60-65.

Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
GIDA,Gor
akhpur
(U.
P)Page31
 1
4.    Wi llar
dHH,merri
ttLL,deanJA,set t
leFA,instr
umental
methodsofanal
ysi
s,Newdel
hi:cbspubl
ishers&di
stri
butor
s.Pageno
-51
3-538.

Tahi
raI
nst
itut
eofMedi
calSc
ienc
es,
GIDA,Gor
akhpur
(U.
P)Page32