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Lec18.Reversed Phase

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  INSTRUMENTAL ANALYSIS AND SEPARATIONSREVERSED PHASE CHROMATOGRAPHY - LECTURE 18Reversed-Phase for No-Io! Aa#$%esReversed-&hase !s %he 'os% o''o %$&e of HPLC( Reversed-&hase o#)'s are r)**ed+ eff! !e%+ re&rod) !,#e+ ad s%a,#e( A)eo)s 'o,!#e &hases are #ess e.&es!ve %ha or*a! &hases+ de%e %!o !s of%e eas!er+ ad #ess %!'e !s re)!red for e)!#!,ra%!o of %he o#)' ,e%/ee r)s( Reversed-&hase hro'a%o*ra&h$ !s s!'!#ar %o %he e.%ra %!o of o'&o)ds fro' /a%er )s!* a or*a! &hase( The s%a%!oar$ &hase+ o''o#$ C-8 or C-18 !s #ess &o#ar %ha %he /a%er-or*a! 'o,!#e &hase+ ad o'&o)ds &ar%!%!o ,e%/ee%he %/o &hases ,ased o %he!r h$dro&ho,! !%$( Po#ar 0h$dro&h$#!  'o#e )#es &ar%!%!o #ess !%o %he s%a%!oar$ &hase ad are e#)%ed f!rs%( No-&o#ar 'o#e )#es are 'ore s%ro*#$ he#d ,$ %he s%a%!oar$ &hase ad are e#)%ed #as%( Med!)' &o#ar!%$ 'o#e )#es are e#)%ed ! %he '!dd#e of %he hro'a%o*ra'+ ad %h)s aa#$%es a ,e se&ara%ed(If &o#ar!%$ /as %he o#$ fa %or !vo#ved+ o'&o)ds /o)#d a#/a$s e#)%e ! %he sa'e order( Ho/ever+ %he re%e%!o of a o'&o)d !s a#so affe %ed ,$ e.&er!'e%a# od!%!os s) h as+ ho! e of %he 'o,!#e &hase+ ho! e of s%a%!oar$ &hase+ ad o#)' %e'&era%)re( So#ve%s for Reversed-Phase Chro'a%o*ra&h$Par%!%!o!* ,e%/ee %he 'o,!#e ad s%a%!oar$ &hases !s ,ased o so#ve%  &ro&er%!es o##e %!ve#$ %er'ed 2so#ve% s%re*%h(3 The %hree fa %ors ,e#!eved %o ,e !vo#ved ! so#ve% s%re*%h are &o#ar!%$+ a !d!%$+ ad ,as! !%$( A re&rese%a%!o of %he s%re*%h of var!o)s so#ve%s !s &rese%ed ! %he so#ve%-se#e %!v!%$ %r!a*#e( I% !s &oss!,#e %o o'&#e%e#$ reverse %he e#)%!o order of so'e o'&o)ds ,$ ha*!* %he so#ve% %$&e(Three /a%er-'!s !,#e so#ve%s+ 'e%hao#+ a e%o!%r!#e ad %e%rah$drof)ra d!ffer os!dera,#$ ! %he!r se#e %!v!%$ &ro&er%!es ad are a#so a e&%a,#e ! %er's of UV a,sor,a e ad v!s os!%$ ad %herefore are %he 'os% o''o#$ )sed or*a! so#ve%s for reversed-&hase se&ara%!os( Me%hao# has %he d!sadva%a*e of for'!* a re#a%!ve#$ v!s o)s so#)%!o /!%h /a%er+ *!v!* h!*h &ress)res+ or re)!r!* #o/er f#o/ ra%es( A e%o!%r!#e !s a e.&es!ve a#%era%!ve %o 'e%hao#( 4)% ! add!%!o %o offer!* d!ffere% se#e %!v!%$+ !% does o% &rod) e %he v!s os!%$ &ro,#e's %ha% 'e%hao# does( Te%rah$drof)ra offers add!%!oa# se#e %!v!%$+ ,)% !%s UV )%off !s re#a%!ve#$ h!*h 0556 '( Co#)' re-e)!#!,ra%!o fo##o/!* a *rad!e% r) !s a#so ') h s#o/er /!%h %e%rah$drof)ra %ha /!%h %he o%her so#ve%s( Ao%her d!sadva%a*es of %e%rah$drof)ra !s%ha% !% !s )s%a,#e ad /he e.&osed %o a!r !% for's h$dro&ero.!des %ha% a rea % /!%h %he sa'&#e+ ad 'a$ &ose a safe%$ ha7ard(Mo,!#e &hases )sed sho)#d o% o%a! #ess %ha 16 or*a! so#ve% /he )s!* s%a%!oar$ &hases s) h as C-18( 9he %he 'o,!#e &hase o%a!s 'ore %ha 16 or*a! so#ve%+ %he a#:ae ha!s are 'ore or #ess e.&aded o)% s%ra!*h% fro' %he s)rfa e( 9!%h Lecture 15 1  h!*her /a%er o%e%+ %he C-18 ha!s o##a&se ad !% !s d!ff! )#% %o ,r!* %he' ,a : %o %he s%ra!*h% ofor'a%!o /he h!*her #eve#s of or*a! so#ve% are added ,a : %o %he 'o,!#e  &hase(The o%her !'&or%a% so#ve% for reversed &hase !s /a%er( H!*h &)r!%$ /a%er 0HPLC *rade !s eeded %o :ee& ,ase#!e o!se %o a '!!')'( ;or 'os% se&ara%!os+ &H o%ro# !s !'&or%a% so a)eo)s ,)ffers are o''o#$ )sed ra%her %ha &)re /a%er( 9he%he 'o,!#e &hase &H !s ear %he &<  a  of a aa#$%e+ %he re%e%!o of %he aa#$%e var!es os!dera,#$ /!%h s'a## ha*es ! &H 0as s'a## as 6(1 )!% ad &ea: sha&es a ,e o'es:e/ed( I% !s %herefore !'&or%a% %ha% %he 'o,!#e &hase !s /e## ,)ffered+ ad %he &H !s a )ra%e#$ 'eas)red( I% !s a#so !'&or%a% %o o%e %ha% %he aa#$%e !%se#f a ha*e %he &H of %he 'o,!#e  &hase /!%h! !%s o e%ra%!o ,ad so o e%ra%!os of %he aa#$%es sho)#d o% ,e %oo #ar*e ad %he sa'&#e &H sho)#d o% ,e %oo d!ffere% fro' %he ,)ffer &H( 4)ffers are #!'!%ed ! %he &H ra*e %ha% %he$ a o%ro# so !% !s ,es% !f %he &<  a  of %he aa#$%e !s s!'!#ar %o %he &<  a  of %he ,)ffer( C!%ra%e ,)ffer has %he adva%a*e of ,e!* a,#e %o s%a,!#!7e &H over a /!de ra*e 05(1-=(>( Th!s !s ,e a)se %he !%ra%e 'o#e )#e has ? !o!7a,#e *ro)&s /!%h d!ffere% &<  a @s( Ho/ever+ ,e a)se of !%s UV a,sor,a e+ !% a o#$ ,e )sed /!%h UV de%e %ors a% /ave#e*%hs a,ove 5?6 '( C!%ra%e has a#so ,ee #a!'ed %o ,e orros!ve %o s%a!#ess s%ee#+ so !f !% !s )sed+ e.%ra are !s eeded %o f#)sh %he s$s%e' af%er a ser!es of r)s( I add!%!o %o %he &<  a  %here are o%her fa %ors !'&or%a% %o ,)ffer se#e %!o( Co'&a%!,!#!%$ /!%h %he de%e %or !s !'&or%a%( Mos% ,)ffers 0o%her %ha !%ra%e a ,e )sed for UV de%e %!o do/ %o /ave#e*%hs of 566 ' or eve #ess( Vo#a%!#e ,)ffers s) h as a''o!)' ar,oa%e+ a''o!)' for'a%e+ a''o!)' a e%a%e+ ad %r!f#)oroa e%! a !d a ,e re'oved ,$ eva&ora%!o ad are o'&a%!,#e /!%h so'e de%e %ors s) h as a #!*h% s a%%er!* de%e %or or 'ass s&e %ro'e%er( I% !s a#so !'&or%a% %ha% %he ,)ffer ,e o'&a%!,#e /!%h %he or*a! &hase ad o% &re !&!%a%e !f h!*h o e%ra%!os of or*a! so#ve% are eeded for %he se&ara%!o( S%a%!oar$ PhasesA#:ae ha!s of 8 0C-8 or 18 0C-18 ar,o a%o's ,oded %o s!#! a are %he 'os% o''o#$ )sed s%a%!oar$ &hases( ;or aa#$ses od) %ed /!%h a h!*h &H 'o,!#e &hase+ a#:aes ,oded %o a &o#$'er s)&&or% are )sed( Po#$'er s)&&or%s are a#so &referred over s!#! a for %he aa#$s!s of ,as! o'&o)ds ,e a)se %he res!d)a# s!#ao# *ro)&s o s!#! a a a)se severe ,ad %a!#!* 0Ne/er %$&e 4 s!#! a a a#so ,e )sed for ,as! aa#$%es(Geera##$ %he #o*er %he a#:ae ha!+ %he #o*er %he re%e%!o( Ho/ever+ re%e%!o !s a#so ,ased o %he a %!v!%$ of res!d)a# s!#ao# *ro)&s( C)rre%#$ ava!#a,#e C-8 ad C-18 o#)'s d!ffer s!*!f! a%#$ ! %he!r re%e%!o &ro&er%!es ad are de&! %ed ! ;!*)re 1( Po#ar s%a%!oar$ &hases s) h as a'!o+ $ao+ ad d!o# a a#so ,e )sed ! reversed &hase hro'a%o*ra&h$( Lecture 15 5  ;!*)re 1( Se#e %!v!%$ &ro&er%!es of C-8 ad C-18 S%a%!oar$ Phases(Effe % of Te'&era%)reI add!%!o %o %he ha*es ! se#e %!v!%$ ,ro)*h% a,o)% ,$ ha*!* %he 'o,!#e ads%a%!oar$ &hases+ o%ro# of %e'&era%)re a a#so ,e )sed %o !'&rove se#e %!v!%$ ad reso#)%!o( I reas!* %e'&era%)re has %he effe % of de reas!* : so %ha% o'&o)ds e#)%eear#!er+ ,)% ha*es ! %e'&era%)re *eera##$ do o% &rod) e #ar*e ha*es ! se#e %!v!%$ for o-!o! so#)%es( Ho/ever+ as %e'&era%)re !s ! reased %he re%e%!o of &#aar o'&o)ds de reases fas%er %ha o-&#aar o'&o)ds res)#%!* ! a ha*e ! se#e %!v!%$( No-A)eo)s Reversed-Phase HPLC;or ver$ h$dro&ho,! sa'&#es %ha% are re%a!ed so s%ro*#$ %ha% %he$ are o% e#)%ed/!%h 166 a e%o!%r!#e+ a o-a)eo)s 'o,!#e &hase a ,e )sed /!%h /ha% /o)#d %$&! a##$ ,e a reversed-&hase o#)' 0C-8+ C-18 e% (( I %h!s ase %he 'o,!#e &hase !s a '!.%)re of 'ore &o#ar ad #ess &o#ar or*a! so#ve%s( T$&! a##$ 'e%hao# or a e%o!%r!#e !s )sed as %he 'ore &o#ar so#ve% 0/ea:er so#ve% for h$dro&ho,! sa'&#es /h!#e h#orofor'+ 'e%h$#ee h#or!de+ 'e%h$#-%-,)%$# e%her+ or %e%rah$drof)ra a o''o#$ )sed as %he #ess &o#ar so#ve% 0s%ro*er so#ve%( The se&ara%!o of aro%eo!ds s) h as α - aro%ee+ β - aro%ee ad #$ o&ee !s a e.a'&#e of a food s !e e )se of o-a)eo)s reversed-&hase hro'a%o*ra&h$( ;or %he se&ara%!o of so'e aro%eo!ds+ #o* ha!s s) h as C-?6 are eeded %o o,%a! ade)a%e reso#)%!o(Chro'a%o*ra&h$ of Io!7a,#e Co'&o)dsI reversed-&hase hro'a%o*ra&h$+ sa'&#e re%e%!o ! reases for 'ore h$dro&ho,! o'&o)ds( 9he a a !d or a ,ase !s !o!7ed 0 over%ed fro' a ) har*ed %o a har*ed 'o#e )#e !% ,e o'es ') h #ess h$dro&ho,! ad /!## e#)%e ') h Lecture 15 ?  ear#!er 0!%s : va#)e a ,e red) ed as ') h as 16-fo#d %o 56-fo#d( A !ds #ose a &ro%o ad ,e o'e !o!7ed 0a )!re a e*a%!ve har*e as &H ! reases( 4ases o %he o%her had+ *a! a &ro%o ad a )!re a &os!%!ve har*e as &H de reases( Therefore+ 0a## o%her %h!*s ,e!* e)a# a !ds /!## e#)%e ear#!er a% h!*her &H ad ,ases /!## e#)%e ear#!er a% #o/  &H( A% !%er'ed!a%e &H+ %he 'o#e )#e !s fo)d &ar%#$ ! %he har*ed for' ad &ar%#$ !%he ) har*ed for'( The &H a% /h! h a o'&o)d !s fo)d e.a %#$ ha#f ! %he har*ed for' ad ha#f ! %he ) har*ed for' !s :o/ as %he &<  a ( ;!*)re 5 sho/s %he re#a%!ve effe % of 'o,!#e &hase &H o %he re%e%!o of a ,as! o'&o)d /!%h a &<  a  of >( A% #o/  &H %he 'o#e )#e !s f)##$ har*ed 0h$dro&h!##!  ad !s o% /e## re%a!ed ! reversed &hase s$s%e's( A% h!*h &H+ %he 'o#e )#e !s o% har*ed 0h$dro&ho&!  ad !s /e## re%a!ed ,$ %he s%a%!oar$ &hase( I% !s eas$ %o see %ha% %he &H of %he 'o,!#e &hase has a &rofo)d effe % o %he re%e%!o of !o!7a,#e o'&o)ds( A#'os% a## of %he &H de&ede% ha*e ! re%e%!o %a:es &#a e 1( &H )!%s o e!%her s!de of %he &<  a  va#)e( A,ove or ,e#o/ %h!s ra*e %he 'o#e )#e !s e!%her har*ed or ) har*ed ad !%s re%e%!o does o% ha*e ') h/!%h ha*es ! &H( So'e 'o#e )#es+ a'!o a !ds are *ood e.a'&#es+ have ,o%h a !d! ad ,as! *ro)&s o %he sa'e 'o#e )#e( Th!s &ro&er%$ !s a##ed a'&ho%er! ( Amphoteric 'o#e )#es have &os!%!ve har*es a% #o/ &H ad e*a%!ve har*es a% h!*h &H( A 'o#e )#e %ha% !s a'&ho%er! !s a##ed a zwitterion ( A% !%er'ed!a%e &H va#)es+ ,o%h %he a !d! ad  ,as! *ro)&s are &ar%!a##$ har*ed( 4e a)se ,o%h *ro)&s are har*ed+ %he 'o#e )#e has !%sh!*hes% !o!7a%!o ad %herefore !%s #o/es% re%e%!o a% !%er'ed!a%e &H(;!*)re 5( Re#a%!ve Re%e%!o for a 4as! Io!7a,#e Co'&o)d a% D!ffere% &H Leve#s(9he aa#$%es are ,as! o'&o)ds+ %he$ a !%era % /!%h %he s!#ao#s of s!#! a- ,ased o#)'s( Th!s a res)#% ! ! rease re%e%!o+ ,ad %a!#!* ad o#)'-%o- o#)' var!a,!#!%$( Therefore o#)'s s&e !f! a##$ des!*ed for se&ara%!* ,ases sho)#d ,e se#e %ed( These o#)'s are 'a)fa %)red %o '!!'!7e %he )',er of ver$ a !d! s!#ao#s( A #o/ &H 'o,!#e &hase a a#so '!!'!7e %he o e%ra%!o of !o!7ed s!#ao#s( S!#ao# effe %s a a#so ,e red) ed ,$ hoos!* ,)ffer a%!os %ha% are s%ro*#$ he#d ,$ %he s!#ao#s ad /!## o% read!#$ e. ha*e /!%h %he aa#$%e !os( A h!*h &H a a#so ,e Lecture 15 > 1234567 pH    R  e   l  a   t   i  v  e   R  e   t  e  n   t   i  o  n   T   i  m  e pK a partially chargedfully chargednot charged
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