EXPERI

MENT:
DEMONSTRATI
ONOFWORKI
NGOFHPLC

Obj
ect
ive
The obj
ect
ivei
sto dr
aw cal
i
brat
ion cur
ve based on r
esul
tsf
ordi
ff
erent
concent
rat
ionsofpur
e(known)compoundandt
hent
oident
if
yandquant
it
atet
hesame
compoundi
nit
ssol
uti
on.

I
ntr
oduct
ion
Thet
erm‘
HPLC’
ori
ginal
l
yst
andsf
or‘
HighPr
essur
eLi
qui
dChr
omat
ogr
aphy
’.The
t
erm‘
Chr
omat
ogr
aphy
’means‘
col
ourwr
it
ing’i
nGr
eek.I
tisbasedonpar
ti
ti
oni
ngof
sol
utesbet
weent
wophases.I
ncol
umnchr
omat
ogr
aphy
,thest
ati
onar
yphasei
sa
hi
ghl
ypor
oussol
i
dmat
eri
alpackedi
nsi
deacy
li
ndr
icalcol
umn(
steelorgl
ass)
,wher
eas
t
hemobi
l
ephasei
sal
i
qui
d,agas,orasuper
cri
ti
calflui
d.I
ncaseofl
i
qui
dasmobi
l
e
phase,t
heoper
ati
oni
ster
medas‘
l
iqui
dchr
omat
ogr
aphy
’.Ast
her
eishi
ghpr
essur
e
gener
ati
on wi
thi
nthe col
umn,i
twas i
nit
ial
l
yter
med as ‘
High Pr
essur
e Li
qui
d
Chr
omat
ogr
aphy
’.Wi
th cont
inued adv
ances i
n per
for
mance [
smal
l
erpar
ti
cles of
st
ati
onar
ysol
i
dphase,ev
enhi
gherpr
essur
e],t
heacr
ony
m HPLC 
remai
nedt
hesame,
butt
henamewaschangedt
o‘Hi
ghPer
for
manceLi
qui
dChr
omat
ogr
aphy
’.Atpr
esent
,
i
t 
isoneoft
hemostpower
fult
ool
sinanal
yti
calchemi
str
yascompoundsi
ntr
ace
concent
rat
ionsasl
owaspar
tspert
ri
ll
i
on[
ppt
]mayeasi
l
ybei
dent
if
ied.I
tcansepar
ate,
i
dent
if
yandquant
it
atet
hecompoundspr
esenti
nanysampl
e,sol
ubl
einal
i
qui
d.HPLC
canbeappl
i
edt
oal
mostanysampl
e,suchasphar
maceut
ical
s,f
ood,nut
raceut
ical
s,
cosmet
ics,
env
ironment
almat
ri
ces,
for
ensi
csampl
es,
andi
ndust
ri
alchemi
cal
s.

Theor
y
I
nHPLC,onephase(
themobi
l
ephase)i
sinconst
antmov
ementr
elat
ivet
othe
ot
herone (
the st
ati
onar
yphase)
.The sampl
e sol
ute mol
ecul
es(
whi
ch ar
eto be
i
dent
if
iedbot
hqual
i
tat
ivel
yandquant
it
ati
vel
y)ar
epar
ti
ti
onedbet
weent
hephases;
t
hosei
nthest
ati
onar
yphasear
eret
ained,wher
eas,t
hosei
nthemobi
l
ephasemov
e.
Adsor
pti
oni
sthebasi
sfort
hei
nter
act
ionbet
weent
hesol
utesandt
hest
ati
onar
yphase.

1
Gener
all
y,asol
utepar
ti
ti
onsbet
weenst
ati
onar
yandmobi
l
ephasesmanyt
housand
t
imes.Di
ff
erentt
ypesofmol
ecul
esspenddi
ff
erentamountoft
imei
nthest
ati
onar
y
phaseandt
hatf
ormst
hebasi
sofsepar
ati
on.Duet
othel
argenumberofpar
ti
ti
oni
ng
st
eps,HPLChasenor
mousr
esol
vi
ngpowerandcansepar
atemi
xtur
esofcomponent
s
wi
thv
erysi
mil
arphy
sical
proper
ti
es.
I
nanal
yti
calchr
omat
ogr
aphy
,iti
sai
medt
ogetquant
it
ati
veand/
orqual
i
tat
ive
i
nfor
mat
ionaboutoneorsev
eralcomponent
sinasampl
emi
xtur
e.Thequal
i
tat
ive
i
nfor
mat
ioni
sobt
ainedf
rom t
her
etent
iont
imesi
nananal
yti
calchr
omat
ogr
am andt
he
quant
it
ati
vei
nfor
mat
ionf
rom t
hear
eas,orhei
ght
,oft
hepeaks.I
nthei
dealcase,t
he
anal
yti
calpeaksar
eGaussi
an;
but
,inr
eal
i
ty,
thepeaksar
eof
tensl
i
ght
lydi
stor
tedwi
tha
smal
ltai
l
.Theadsor
pti
oni
sot
her
msr
elat
ethemobi
l
e-phaseconcent
rat
ionwi
tht
he
st
ati
onar
y-phaseconcent
rat
ion.Thi
srel
ati
oni
sli
neari
nanal
yti
calchr
omat
ogr
aphyasi
t
i
sper
for
med atl
ow concent
rat
ionscor
respondi
ng t
othe i
nit
ial(
pract
ical
l
yli
near
)
sect
ionoft
headsor
pti
oni
sot
her
m.Becauseoft
his,anal
yti
calchr
omat
ogr
aphyi
sal
so
cal
l
edl
i
nearchr
omat
ogr
aphy
.
Basi
cRet
ent
ionModel
sandKi
net
ics
A sampl
emi
xtur
eappl
i
ed ont
het
op ofachr
omat
ogr
aphi
ccol
umnwi
l
lbe
t
ranspor
tedt
hrought
hecol
umnbyt
hemobi
l
ephaseandi
napr
oper
lydesi
gnedcol
umn,
t
hesol
uteswi
l
lbeel
utedatt
hecol
umnout
letassepar
atezones.Thesol
utemi
grat
ion
r
atei
ssi
mpl
y(di
stance/
ti
me)
.Ev
eni
fthesol
utezonemi
grat
esataconst
antr
ate
t
hrought
hecol
umnbed,att
hemol
ecul
arl
evel
,thei
ndi
vi
dualmol
ecul
esdoa“
random
wal
k.”Mor
especi
fical
l
y,asi
ngl
emol
ecul
etr
anspor
tedbyt
hemobi
l
ephasei
sadsor
bed
f
oracer
tai
nti
meandi
nthenextmomentdesor
bed,adsor
bedagai
n,andsooni
n
t
housandsofst
epsdur
ingi
tst
rav
elal
ongt
hecol
umn.Onanav
erage,t
hei
ndi
vi
dual
mol
ecul
ewi
l
lbei
nthemobi
l
ephasedur
ingt
het
ime‘
tm’andi
nthest
ati
onar
yphase
dur
ingt
het
ime‘
ts’
.I
tst
otalr
esi
dencet
imei
nthecol
umnbecomes‘
tm+t
s’
.Amol
ecul
eis
adsor
bedf
oranav
eraget
imeof‘
ts’
,whi
l
eiti
sdesor
bedf
oranav
eraget
imeof‘
tm’
,and
i
tthenmi
grat
eswi
tht
hev
eloci
tyoft
hemobi
l
ephase,
‘ux’
.Thi
smeanst
hatt
hemol
ecul
e
wi
l
lspendaf
ract
ion,for‘
tm/
(tm +t
s)’ofi
tst
imemov
ingwi
th.Thev
eloci
tyoft
he
mol
ecul
e,
us=ux.
f

2
 Shor
tl
yaf
tert
hesampl
est
art
stot
rav
el,
thedi
str
ibut
ionequi
l
ibr
ium oft
hesol
ute
bet
weent
hest
ati
onar
yphaseandt
hemobi
l
ephasei
sest
abl
i
shed.I
fiti
sassumedt
hat
sol
utei
nal
i
mit
edpar
toft
hecol
umnr
epr
esent
sthesol
utemol
ecul
es’
aver
agebehav
ior
,
acer
tai
namountofsol
utei
sint
hest
ati
onar
yphase,Qs,andacer
tai
namounti
sint
he
mobi
l
ephase,
Qm.Thet
otalamounti
nthezonewi
l
lbe(
Qm +Qs)andt
hef
ract
ionoft
he
sol
utei
nthemobi
l
ephasei
s,Qm/
(Qm +Qs)
.Ther
emustbeadi
rectcor
rel
ati
onbet
ween
t
heamounti
nthezonesi
nthemobi
l
ephaseandt
hecor
respondi
ngt
imef
ract
ionf
ort
he
sol
utei
nthemobi
l
ephase.Thel
argerf
ract
ionoft
imet
hesol
utespendsi
nthemobi
l
e
phase,acor
respondi
ngl
ylar
gerquant
it
yofsol
utei
spr
esenti
nthemobi
l
ephase.Thi
s
gi
ves:
us=ux.Qm/
(Qm +Qs)=ux/
(1+k)
wher
e,k=Qs/
Qm
Her
e,‘
k’i
s‘Ret
ent
ionFact
or’
andi
sdef
inedast
her
ati
oof‘
tot
alamountofsol
utei
nthe
st
ati
onar
yphase’
and‘
tot
alamountofsol
utei
nthemobi
l
ephase’
i.
e.,
k=(
Cs.
Vs)
/(Cm.
Vm)
wher
e,‘
C’i
sconcent
rat
iont
erm and‘
V’i
svol
umet
erm.
Fr
om chr
omat
ogr
am,
‘k’
canbedet
ermi
nedas(
tR–t
0)/
t0

wher
e,t
Rist
her
etent
iont
imef
ort
hespeci
fi
ccompound(
insol
ute)i
.e.
,el
apsedt
ime
bet
weent
hemomentofi
nject
ion(
ti
mezer
o)andt
het
imewhent
hepeakmaxi
mum
el
utesand t
0ist
het
rav
elt
imef
ort
hemobi
l
ephaseort
her
etent
iont
imeofan
unr
etai
nedcomponent
.

Wor
kingPr
inci
ple
Thecomponent
sofabasi
cHPLCsy
stem ar
eshowni
nthesi
mpl
edi
agr
am i
nFi
g.
1.Ar
eser
voi
rhol
dst
hesol
ventormobi
l
ephase.Ahi
gh-
pressur
epump(
sol
ventdel
i
ver
y
sy
stem orsol
ventmanager
)isusedt
ogener
ateandmet
eraspeci
fi
edf
low r
ateof
mobi
l
e phase,t
ypi
cal
l
y mi
l
li
li
ter
s per mi
nut
e.An i
nject
or (
sampl
e manager or
aut
osampl
er)i
sabl
etoi
ntr
oduceori
njectt
hesampl
eint
othecont
inuousl
yfl
owi
ng
mobi
l
ephasest
ream t
hatcar
ri
est
hesampl
eint
otheHPLC col
umn.Thecol
umn
cont
ainst
hechr
omat
ogr
aphi
cpacki
ngmat
eri
alneededt
oef
fectt
hesepar
ati
on.Thi
s
packi
ngmat
eri
ali
scal
l
edt
hest
ati
onar
yphasebecausei
tishel
dinpl
acebyt
hecol
umn

3
har
dwar
e.Ast
hesepar
atedcompoundsl
eav
ethecol
umn,t
hesepassi
mmedi
atel
yint
o
t
hedet
ect
or.Thedet
ect
orcont
ainsaf
lowcel
lthatdet
ect
seachsepar
atedcompound
bandagai
nstabackgr
oundofmobi
l
ephase.Anappr
opr
iat
edet
ect
orhast
heabi
l
ityt
o
senset
hepr
esenceofacompoundandsendi
tscor
respondi
ngel
ect
ri
calsi
gnalt
oa
comput
erdat
ast
ati
on.Achoi
cei
stobemadeamongmanydi
ff
erentt
ypesofdet
ect
ors,
dependi
ngupont
hechar
act
eri
sti
csandconcent
rat
ionsoft
hecompoundst
hatneedt
o
besepar
atedandanal
yzed.Themobi
l
ephaseexi
tst
hedet
ect
orandcanbesentt
o
wast
e,orcol
l
ect
ed,as desi
red.Hi
gh-
pressur
etubi
ng and f
it
ti
ngs ar
e used t
o
i
nter
connectt
hepump,i
nject
or,col
umn,anddet
ect
orcomponent
stof
ormt
hecondui
t
f
ort
hemobi
l
ephase,
sampl
e,andsepar
atedcompoundbands.
Thedet
ect
ori
swi
redt
othecomput
erdat
ast
ati
on,
theHPLCsy
stem component
t
hatr
ecor
dst
heel
ect
ri
calsi
gnalneededt
ogener
atet
hechr
omat
ogr
am oni
tsdi
spl
ay
andt
oident
if
yandquant
it
atet
heconcent
rat
ionoft
hesampl
econst
it
uent
s.Si
nce
sampl
ecompoundchar
act
eri
sti
cscandi
ff
erwi
del
y,sev
eralt
ypesofdet
ect
orshav
e
beendev
eloped.Forexampl
e,i
facompoundcanabsor
bul
tr
avi
olet(
UV)l
i
ght
,aUV-
absor
bancedet
ect
ori
sused.I
fthecompoundf
luor
esces,af
luor
escencedet
ect
ori
s
used.I
fthecompounddoesnothav
eei
theroft
hesechar
act
eri
sti
cs,amor
euni
ver
sal
t
ypeofdet
ect
ori
sused,suchasanev
apor
ati
ve-
li
ght
-scat
ter
ingdet
ect
or(
ELSD)or
r
efr
act
ivei
ndex(
RI)det
ect
or.Themostpower
fulappr
oachi
stheusemul
ti
pledet
ect
ors
i
nser
ies.Forexampl
e,aUVand/
orELSDdet
ect
ormaybeusedi
ncombi
nat
ionwi
tha
massspect
romet
er(
MS)t
oanal
yzet
her
esul
tsoft
hechr
omat
ogr
aphi
csepar
ati
on.Thi
s
pr
ovi
des,f
rom asi
ngl
einj
ect
ion,mor
ecompr
ehensi
vei
nfor
mat
ionaboutananal
yte.
Thepr
act
iceofcoupl
i
ngamassspect
romet
ert
oanHPLCsy
stem i
scal
l
edLC/
MS.

4
 Fi
g.1:
High-
Per
for
manceLi
qui
dChr
omat
ogr
aphy(
HPLC)Sy
stem.
 

Exper
iment
alPr
ocedur
e
1. Swi
tchonbot
hpumps(
meantf
orpumpi
ngsol
vent
sAandB)
.
2. Swi
tchoni
nter
face.
3. Swi
tch on appr
opr
iat
e det
ect
or(
dualabsor
bance UV-
Vis orRI
)and al
l
ow
suf
fi
cientt
ime (
usual
l
y 5 mi
nut
es)t
o war
m up.I
twi
l
lshow ‘
OK’f
oral
l
component
swheni
tbecomes‘
Ready
’.
4. Swi
tchont
hecomput
erat
tachedwi
tht
hissy
stem.
5. Pr
imepumpsonebyoneunt
ilt
her
eisnobubbl
ecomi
ngi
nthel
i
qui
dtakenout
f
rom t
hepump.
6. Pur
gepumpsonebyonebygi
vi
ngi
nst
ruct
ioni
nar
emot
emanner
.
7. Opent
he‘
Empower
’sof
twar
e.Cl
i
ck‘
BrowsePr
oject
s’.Choosesui
tabl
emet
hod
f
rom av
ail
abl
e ones (
Ifnecessar
y,dev
elop new met
hod)
.Choose sui
tabl
e
det
ect
or.Choosesol
vent(
mobi
l
ephase)el
uti
onopt
ion.

5
8. Di
ssol
vepur
ecompoundi
nasui
tabl
esol
venti
ndi
ff
erentpr
opor
ti
onst
oget
sol
uti
onsofdi
ff
erentconcent
rat
ions.Pr
epar
esol
uti
on ofsampl
econt
aini
ng
unknowncompoundi
nsamesol
vent
.
9. I
njectonesol
uti
onofpur
ecompoundbymi
crosy
ringei
nthe‘
I
nject
ionPor
t’unt
il
excesssol
uti
oncomesoutandgetcol
l
ect
edi
nthe‘
Col
l
ect
or’
.Qui
ckl
y,change
t
hemodeof‘
I
nject
ionPor
t’f
rom ‘
Load’t
o‘I
nj’
.Si
mul
taneousl
y,pr
ess‘
Runt
he
sampl
es’
opt
ioni
n‘Empower
’.
10. Repeatt
heabov
est
epf
orot
hersol
uti
onsofpur
ecompound.
11. Forknownconcent
rat
ionsofpur
ecompound,
not
edownar
eaundert
hepeakf
or
eachconcent
rat
ion.
12. Runt
hesampl
eundersamecondi
ti
on.Obser
vewhet
heranypeaki
scomi
ngat
t
hesamet
imeasobser
vedi
ncaseofpur
ecompound.I
fsuchapeakcomes,
not
edownar
eaundert
hepeak.I
fnecessar
y(f
orshi
ft
ingofbasel
i
nedur
ing
gener
ati
onofpeak)
,manual
l
yint
egr
atet
hepeakandnot
edownar
eaaf
tersuch
manual
int
egr
ati
on.

Resul
tsandCal
cul
ati
on
1. Not
edownt
her
esul
tsf
orknownconcent
rat
ionsf
orpur
ecompound i
nthe
f
oll
owi
ngt
abl
e:
Tabl
e1:r
esul
tsf
orknownconcent
rat
ionsofpur
ecompound
RunNo. Concent
rat
ionof Peakar
ea,
A
purecompound,C
(mg/ml) (
µV.
sec)

Ont
hebasi
sofr
esul
tsshowni
nabov
etabl
e,dr
awacal
i
brat
ion(
Cvs.A)cur
ve

6
 (
thi
scur
veshoul
dbeast
rai
ghtl
i
nepassi
ngt
hroughor
igi
nact
ual
l
y).

2. Not
edownar
eaundert
hepeak(
ifi
tcomesatt
hesamet
imeasobser
vedi
ncase
ofpur
ecompound)
.Poi
ntoutt
hisar
ea(
denot
ed asY)i
n‘A’axi
s;gett
he
cor
respondi
ngconcent
rat
ion(
denot
edasX)f
rom cal
i
brat
ioncur
ve(
asshowni
n
Fi
g.2)
.
A

  Y

▪
▪

X C
Fi
g.2:
Repr
esent
ati
vedi
agr
am f
orcal
i
brat
ioncur
ve(
andsi
mul
taneousi
dent
if
icat
ionand
quant
it
ati
onofacompoundi
nit
ssol
uti
on)
.

Pr
ecaut
ions
1. Ensur
ethatt
her
eisnobubbl
einmobi
l
ephasebysoni
cat
ionf
orasuf
fi
cientt
ime
i
nter
val
.
2. Fi
l
tersampl
epr
oper
lysucht
hatt
her
eremai
nsnopar
ti
clewhi
chcancl
ogt
he
chr
omat
ogr
aphi
ccol
umn.

ANNEXURE(
notr
equi
redf
orl
abcopy
)
 Det
ect
orsi
nPr
esentSy
stem
DualAbsor
banceUl
tr
avi
olet
-Vi
sibl
eDet
ect
or
Thedet
ect
oroper
atesi
nsi
ngl
ewav
elengt
hmodebydef
aul
t.Det
ect
orsuppor
ts
moni
tor
ingofasi
ngl
ewav
elengt
hfr
om 190nm t
o700nm,
set
tabl
ein1-
nm i
ncr
ement
s.
I
nthedualwav
elengt
hmode,
thedet
ect
orhasmor
ecapabi
l
ityt
hanasi
ngl
ewav
elengt
h
det
ect
orf
ori
ncr
easi
ngsensi
ti
vi
tywi
thMaxPl
otandqual
i
tat
ivei
nfor
mat
ionf
rom Rat
io

7
Pl
ot.
MaxPl
ot:The absor
bance spect
ra ofdi
ff
erentcompounds hav
e di
ff
erentl
ambda
maxi
ma.Thegr
eat
estabsor
bancewi
l
lbeobt
ainedatt
hel
ambdamaxi
mum f
oreach
compound. MaxPl
oti
sadualwav
elengt
hfeat
uret
o maxi
mizesensi
ti
vi
tyatt
wo
wav
elengt
hsi
nasi
ngl
echr
omat
ogr
am.Theuserchoosest
wowav
elengt
hs,onef
or
eachchannelandset
stheout
putofonechannelf
orMaxPl
ot.Ast
hedet
ect
ormoni
tor
s
a chr
omat
ogr
am,t
he hi
ghestabsor
bance f
rom ei
therwav
elengt
his out
put
ted as
MaxPl
ot.Thef
igur
ebel
owi
l
lust
rat
est
hisf
eat
ure.TheMaxPl
oti
smor
esensi
ti
vet
han
ei
theroft
hepeakatanyonewav
elengt
h.
Rat
ioPl
ot:TheDet
ect
orcanpl
ott
her
ati
oofpeaksatt
wowav
elengt
hst
hatar
echosen
byt
heuseront
wochannel
s.Thet
wowav
elengt
hsar
etwopoi
ntsont
hecompounds'
spect
ra. Whent
heRat
ioPl
oti
sfl
atatt
het
op,t
her
ati
ooft
het
woabsor
bancesi
s
const
ant
.Thi
sconst
antr
ati
osuggest
speakhomogenei
ty.

Ref
ract
iveI
ndex(
RI)Det
ect
or
Maj
oradv
ant
ageofr
efr
act
ivei
ndex(
RI)det
ect
orl
i
esi
ntheuni
ver
salnat
ureofi
ts
r
esponse.I
tis wel
l
-sui
ted t
o anal
yze compounds t
hatar
e wi
thoutst
rong UV
chr
omophor
es,f
luor
ophor
es,orel
ect
rochemi
calori
oni
cact
ivi
ty.RIdet
ect
orsar
eused
t
oanal
yzecar
bohy
drat
esandl
i
pidsandi
npol
ymeranal
ysesbygel
-per
meat
ionorsi
ze-
excl
usi
on chr
omat
ogr
aphy
.RIdet
ect
ors wor
k by measur
ing changes i
nthe bul
k
pr
oper
ti
es of t
he sol
vent and t
he sol
utes under i
nvest
igat
ion,expl
oit
ing t
he
i
mpr
obabi
l
ityt
hatsol
vent
sandsol
uteswoul
dshar
ethei
dent
icalr
efr
act
ivei
ndex.RI
det
ect
ioni
sinher
ent
lyl
esssensi
ti
vet
hanot
herdet
ect
ionmet
hods.I
tisnotsui
tabl
eto
gr
adi
entanal
yses,
andchangesi
nsol
ventt
emper
atur
eandpr
essur
ecancausebasel
i
ne
i
nst
abi
l
ity
.Al
so,t
hepossi
bil
i
tyexi
stst
hatposi
ti
veornegat
ivepeakscanappeardur
ing
asi
ngl
eanal
ysi
s.

 Chr
omat
ogr
am
Achr
omat
ogr
am i
sar
epr
esent
ati
onoft
hesepar
ati
ont
hathaschemi
cal
l
y
(
chr
omat
ogr
aphi
cal
l
y)occur
redi
ntheHPLC sy
stem.A ser
iesofpeaksr
isi
ng
f
rom abasel
i
nei
sdr
awnonat
imeaxi
s.Eachpeakr
epr
esent
sthedet
ect
or

8
 r
esponsef
oronepar
ti
cul
arcompound.Thechr
omat
ogr
am i
spl
ott
edbyt
he
comput
erdat
ast
ati
on.
 

Fi
g.2:
Creat
ionofPeaksi
nChr
omat
ogr
am.

I
dent
if
yingandQuant
it
ati
ngCompounds
Eachcompoundi
nsampl
emi
xtur
eel
utesataspeci
fi
clocat
ion,measur
edbyt
he
r
etent
iont
ime(
tR)
.Bycompar
ingeachpeak’
sret
ent
iont
ime(
tR)wi
tht
hatofi
nject
ed
r
efer
encest
andar
dsi
nthesamechr
omat
ogr
aphi
csy
stem (
mobi
l
ephase,i
tsf
lowr
ate,
st
ati
onar
yphase,det
ect
orandi
tscondi
ti
onsr
emai
nsame)
,eachcompoundcanbe
i
dent
if
ied.
 
Int
hechr
omat
ogr
am showni
nFi
g.3,
pur
est
andar
dcompound(
her
e,acr
ylami
de
asexampl
e)hasel
utedf
rom t
hecol
umnat2.
85mi
nut
esundercer
tai
ncondi
ti
onsoft
he
chr
omat
ogr
aphi
csy
stem.Whenanew sampl
eisi
nject
edi
ntot
hechr
omat
ogr
aphi
c
sy
stem r
unni
ngundert
hesamecondi
ti
ons,apeakobser
vedat2.
85mi
nut
escl
ari
fi
es
t
hatt
hesampl
econt
ainst
hesamecompound(
acr
ylami
de)andt
hisi
sshowni
nFi
g.4.

9
 Fi
g.3:
Chr
omat
ogr
am ofAPur
eCompound(
Standar
d).
 
Oncei
dent
it
yisest
abl
i
shed,t
henconcent
rat
ionoft
hecompoundi
nthesampl
e
i
stobedet
ermi
nedf
rom t
hechr
omat
ogr
am andt
her
elat
eddat
afr
om t
hedet
ect
or.The
det
ect
orbasi
cal
l
yrespondst
otheconcent
rat
ionoft
hecompoundbandasi
tpasses
t
hrought
hef
low cel
l
.Themor
econcent
rat
edi
tis,t
hest
rongert
hesi
gnal
;gr
eat
eri
s
peak hei
ghtabov
ethe basel
i
ne.Ar
ea undert
he cur
ve(
fort
he peak)i
s di
rect
ly
pr
opor
ti
onal
tot
heconcent
rat
ionofcompound.
 

10
 Fi
g.4:
Ident
if
icat
ionandQuant
it
ati
onofACompoundPr
esenti
nASampl
e.

 Ty
pesofmobi
l
ephaseel
uti
on
 I
socr
ati
cEl
uti
on:I
nthi
smode,composi
ti
onoft
hemobi
l
ephase,ei
therapur
e
sol
ventorami
xtur
e,r
emai
nst
hesamet
hroughoutt
her
un.
 Gr
adi
entEl
uti
on:I
nthi
smode,composi
ti
onoft
hemobi
l
ephasecomposi
ti
on
changes dur
ing t un.Thi
he r s mode i
s usef
ulf
orsampl
es t
hatcont
ain
compounds t
hatspan a wi
de r
ange ofchr
omat
ogr
aphi
c pol
ari
ty.As t
he
separ
ati
onpr
oceeds,t
heel
uti
onst
rengt
hoft
hemobi
l
ephasei
sincr
easedt
o
el
utet
hemor
est
rongl
yret
ainedsampl
ecomponent
s.

 Ty
pesofHPLCoper
ati
on
 Nor
mal
phaseHPLC:
Itusesanon-
pol
armobi
l
ephaseandpol
arst
ati
onar
yphase.
 Rev
ersephaseHPLC:I
tusesapol
armobi
l
ephaseandnon-
pol
arst
ati
onar
y
phase.
Pr
esent
ly,r
ever
sed-
phasechr
omat
ogr
aphyi
susedf
orappr
oxi
mat
ely75% ofal
lHPLC
met
hodsbecausei
tismor
erepr
oduci
bleandhasbr
oadappl
i
cabi
l
ity
.Mostoft
hese
pr
otocol
suseast
hemobi
l
ephaseanaqueousbl
endofwat
erwi
thami
sci
ble,pol
ar
or
gani
csol
vent
,suchasacet
oni
tr
il
eormet
hanol
.Thi
sty
pical
l
yensur
est
hepr
oper
i
nter
act
ionofanal
yteswi
tht
henon-
pol
ar,hy
drophobi
cpar
ti
clesur
face.AC18–bonded
si
l
ica[
somet
imescal
l
edODS]i
sthemostpopul
art
ypeofr
ever
sed-
phaseHPLCpacki
ng.

11