r|nc|p|es

of
chromatography
repared by
urvashl pandey
Shallesh slngh
GUIDLD 8
SAUkA8n ANDL
SimpIified Definifion:
Chromofogrophy seporofes fhe componenfs of o
mixfure by fheir disfincfive offrocfion fo fhe mobiIe
phose ond fhe sfofionory phose.
ExpIonofion:
Compound is pIoced on sfofionory phose
MobiIe phose posses fhrough fhe sfofionory phose
MobiIe phose soIubiIi;es fhe componenfs
MobiIe phose corries fhe individuoI componenfs o
cerfoin disfonce fhrough fhe sfofionory phose,
depending on fheir offrocfion fo bofh of fhe
phoses
Def|n|t|on of Chromatography
@LkMINCLCG
ANAL@L he analye ls a subsance be separaed durlng
chragraphy
S@A@ICNAk nASL he S ls a subsance whlch ls flxed ln
he place fr he chragraphy prcedure eg slllca layer ln
1LC
MC8ILL nASL ls a phase whlch a ves ln a deflnle
dlrecln l ay be a llquld (LC) gas (CC) 1he blle phase
cnsls f he saple belng separaed and he slven ha
ves he saple hrugh he clun
LLUA@L he eluae ls he blle phase leavlng he clun
LLULN@ he eluen ls he slven ha wlll carry he analye
Cont
SCLU@Lrefers he saple cpnens ln parlln
chragraphy
SCLVLN@ refers any subsance capable and slubllslng
her subsance and especlally he llquld blle phase ln LC
LLU@ICN recvery f he separaed subsances by a
cnlnuus flw f he blle phase
Chragraphy sae as exracln
a) Cne phase held ln place
salnary phase
slld aerlal (packlng aerlal)
Anher phase fluld phase
blle phase
exaaple gas (CC)
llquld (LC)
8as|c pr|nc|p|e of chromatography
b
Cont|nued
W 1he basls f all frs f chragraphy ls he
parlln cefflclen (k
d
) whlch descrlbes he way ln
whlch a cpund dlsrlbues lself beween w
llsclble phases
W (Cncenraln ln slven A)/(cncenraln ln
slven 8) k
d
W 1hse subsances dlsrlbued preferenlally ln he
vlng phase passes hrugh he chragraphlc
syse faser han hse dlsrlbued preferenlally ln
he salnary phase
W As a cnsequence he subsances are elued fr
he clun ln lnverse rder f here dlsrlbuln
cefflclens wlh respec he salnary phase
kLMLM8Lk
W 1he salnary phase ls CLAk
W 1he re plar cpnen lneracs re
srngly wlh he salnary phase
W 1he re plar cpnen ves mote
slowly
W 1he nnplar cpnen ves mote
toplJly
o|ar|ty of So|vents
W Water Acet|c Ac|d
W Lthy|eneg|yco|
W Methano|
W Lthano|
W Isopropano|
W yr|d|ne
W Aceton|tr||e
W N|tromethane
W D|ehy|am|ne
W An|||ne
W D|methy|su|fox|de
W Lthy|acetate
W D|oxane
W Acetone
W D|cho|oroethane
W @etrahydrofuran
W D|cho|oromethane
W Ch|oroform
W D|ethy|ether
W 8enzene
W @o|uene
W ky|ene
W Carbontetrach|or|de
W Cyc|ohexane
W etro|eum ether nexane
W entane NCN CLAk
W
CLAk
So|vent system for paper chromatography
S M
waer lsprpanl anla waer (912)
waer nbuanl acelc acldwaer (413)
waer phenl sauraed wlh waer
fralde chlrfr /benzene/ benzenecyclhexane
So|vent system for @LC
S M
slllca gel chlrfr penanl acelc acld (30301)
Cellulse ehanl chlrfr cnc anla sl
L|qu|d ||qu|d chromatography
S M
waer chlrfr / carbn erachlrlde
buanl / lsacene / acene hcl
ehanl / buanl CPCL
3
Co|umn part|t|on chromatography
S M
Waer alchl(nbuanl lsbuanl)
Waer + acld hydrcarbns(benzene
hexane)
Waer + alkallne
Waer buffer cpnens CPCL
3
Aqueus alchls (ehyl alchl) ehyl aceae
keversed phase chromatography
S M
nbuanl waer
canl waer + acld
Chlrfr waer + alkall
nLC
S M
Waer rganlc slven packed cluns wlh
subslued sllxanes
@ypes of chromatography
CN @nL 8ASIS CI MLCnANISM
1 AuSC81lCn
2A81l1lCn
3 Alllnl1?
4 lCn LxCPAnCL
3 CLL L8MLA1lCn
plar sp
fr CC LC fr CC
reslnSC
3

gel fllraln
reslnn(CP
3
)
3
+
by slze
4sk Yourse/f 204 p4l2
pP and lnlc srengh
Most se|ect|ve one
CN @nL 8ASIS CI S@A@ICNAk nASL
1slld llquld
2slldgas
3llquldllquld
4llquldgas
CN @nL 8ASIS CI MC8ILL nASL
1llquld chragraphy
2gas chragraphy
CN @nL 8ASIS CI USLS
1reperalve
2analylcal purpses
CN @nL 8ASIS CI NA@UkL CI S@A@ICNAk nASL
1NCkMAL nASL ln hls blle phase and he saple are
rganlc and usually he salnary phase ls plar ln hls rganlc
saples are analysed
kLVLkSL nASL ln hls he blle phase ls plar such as waer
and he salnary phase ls nn plar
eg S ( nbuanl chlrfrcanl)
M(waer waer +alkallalchls)
aper chromatography
1hls echnlque ls a ype f parlln chragraphy ln whlch he
subsances are dlsrlbued beween w llquld S (usually waer)
whlch ls held ln he flbres f papers and called as salnary phase
M ls a llquld
nLC
Llquld chragraphy ls n wldely used as eans f separalng
rganlc cpund hls prble was verce by PLC ln hls he
pressure ls applled uslng a puplng syse fr frclng he M a
uch hlgher rae whlch prvldes rellable flw raes Lluln pwer
was lprved due he lnrducln f PLC
W Cont|nued
W 1he chlce f sulable blle phase ls vlal ln PLC
W 1he elulng pwer f blle phase ls deerlned by ls
verall plarly plarly f salnary phase and he naure f
saple
W lr nral phase separalns elulng pwer lncreases wlh
lncreaslng plarly f he slven
W lr reverse phase separalns elulng pwer decreases wlh
lncreaslng plarly
W 1he cn slvens used ln PLC are flaable and se
are xlc s PLC lnsruenaln shuld be used well
venllaed labs
Isocrat|c e|ut|on
W ln hls echnlque slngle slven lxure ls used fr he whle
analysls
W l ls relalvely used fr slple separalns
W 1hls ls a slpler echnlque han gradlen eluln ehd
W 1he requlred slven syse wlll prbably cnaln re han
ne cpnen
W l ls bes descrlbed by drawlng a slven rlangle fr ernary
syses and erahedrn fr quarernary cpslln
W 1he cpslln f any lxure ls hen glven by any pln
wlhln he rlangle r erahedrn
W Grad|ent e|ut|on
W 1hls echnlque can frequenly separae slues ha cann be
separaed by lscralc peralns even wlh lxed slvens
W l ls especlally effeclve where he saple cpnens dlffer
wldely ln plarly
W Lw pressure lxlng syses are used
W 1he slvens fr separae reservlrs are fed a lxlng
chaber and hen lxed slven ls hen puped he
clun
W l lprves he resluln f chragra
W l lncreases he sensllvly
@LC
1LC ls slllar paper chragraphy excep ha a hln
layer f se lner aerlal he chlce f bes slven ls
ne f he s lpran declsln ln 1LC
So|vent se|ect|on
1he chlce f bes develplng slven ls ne f he s
lpran declsln ln 1LC
Wlf naure f cpnen ls n knwn he bes eluen ls
fund by rlal and errr uslng sall very rapld runnlng
1LC plaes
Stah|'s tr|ang|e
lf he chelcal naure f he slue be separaed ls knwn l ls
psslble knw a sulable slven uslng sahl's rlangle
Wl ls lnerrelaed wlh adsrben aclvly naure f slue and naure
f slven
Wlf he rlangle ls raed he plns M S L respeclvely deerlnes
he ype f lxure be separaed aclvly f he adsrben and
he plu plarly f he eluen
W1he apprprlae cndlln fr gd separaln are deerlned by
ralng he rlangle
W1he angles f he rlangle crrespnds he prperles f he
saple salnary phase and blle phase
L|qu|d chromatography
1he relalve aracln f slue specles beween he salnary
phase and blle phase ls represened by parlln cefflclen
W1he cpnens f a lxure are separaed lf here parlln
cefflclen beween he slven are sufflclen dlfferen
Wlf he saple ls parlally sluble ln w llsclble slvens he
saple dlsrlbues lself beween hese slvens wlh x

and x
s

W k
x
(x
s
)/(x

)
Wlr hls we can als deerlne dlsrlbuln cefflclen (k)
Wlf k
x
ls hlgh he saple ls sly ln salnary phase vlce versa
Wlf vlue f blle and salnary phase ls lncluded ln calculaln f
dlsrlbuln hen capacly facr (k
x
)
Wk
x'
(al les f x ln salnary phase)/(al les f x ln blle
phase)
Pw d we descrlbe a
chragra 1
1) Chromatogram
A graph show|ng the detectors
response as a funct|on of e|ut|on
t|me
band's shapes pos|t|on reso|ut|on
Pw d we descrlbe a
chragra
Pw d we descrlbe a
chragra
8ase||ne he base llne ls any par f chragra where nly
blle phase ls eerglng fr he clun
eak max|mum he peak axlu ls he hlghes pln f he
peak
In[ect|on po|nt ls ha pln r le when he saple ls placed
n he clun
Dead po|nt ls he pslln f he peak axlu f an
unrealned slue
Dead t|me(t
o
) ls he le elapsed b/w he ln[ecln pln and
he dead pln
Dead vo|ume (v
o
) ls he vlue f he blle phase passed
hrugh he clun b/w he ln[ecln pln and he dead pln
ketent|on t|me(t
r
) ls he le elapsed b/w he ln[ecln pln
and he peak axlu Lach slue wlll have a characerlslc reenln
le
ketent|on vo|ume(v
r
) ls he vlue f blle phase hrugh
he clun b/w he ln[ecln pln and he peak axlu
Corrected retent|on t|me(t'
r
) ls he le elapsed b/w he
dead pln and he peak axlu
Corrected retent|on vo|ume (v'
r
) ls he vlue f blle
phase passed hrugh he clun b/w he dead pln and he peak
axlu l wlll als be he reenln vlue he dead vlue
eak he|ght (h) ls he dlsance b/w he peak axlu and he
base llne geerlcally prduced beneah he peak
eak w|dth(w) ls he dlsance b/w each slde f he peak
eak w|dth at ha|f he|ght ls he dlsance b/w each slde f a
peak easured a half he peak helgh 1he peak wldh easure a
half helgh has n slgnlflcance wlh respec chragraphy
hery
eak w|dth at the base ls he dlsance b/w he lnersecln
f he angens drawn he slde f he peak and he peak base