Experiment 2: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC): METHOD DEELOPMENT
D!te "# Experiment: 2$%&%2$'
Gr"p: A*2&+ ,D'
Le-trer: •
DR MARDIANA *AAID
•
PROF .URAIDAH A/DULLAH MUNIR
N!me: AMAR AMAR *AF0AN *AF0AN /IN /IN MOHD ALI ALI HANAPIAH
*t1ent ID: 2$'+22222
Gr"p mem3er4: •
•
*ALMA I.ATI I.ATI /INTI *INAR MA*HURI (2$'+5,65) NURUL *HA.REENA /INTI .UL7AFLY .UL7AFLY (2$'+2+52&)
Experiment 2: Hi89 Per#"rm!n-e Lii1 C9r"m!t"8r!p9; (HPLC): Met9"1 De
Objective
T" "ptimi>e ! 4ep!r!ti"n "# ! mixtre "# + -"mp"n14 4in8 HPLC 3;
Introduction
Hi89 Hi89 per#"r per#"rm!n m!n-e -e =ii1 =ii1 -9r"m!t -9r"m!t"8r "8r!p9; !p9; i4 t9e m"4t ?i1e=; ?i1e=; 4e1 4e1 "# !== "# t9e !n!=;ti-! !n!=;ti-!== 4ep!r!ti"n te-9nie@ It4 4it!3=e #"r 4ep!r!tin8 n"n<"=!ti=e 4pe-ie4 "r t9erm!==; #r!8i=e "ne4@ P!rtiti"n -9r"m!t"8r!p9; i4 t9e m"4t ?i1e=; 4e1 "# !== t9e #"r t;pe4 "# =ii1 -9r"m!t"8r!p9; pr"-e1re@
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O#ten in -9""4in8 ! -"=mn #"r ! p!rtiti"n -9r"m!t"8r!p9i- 4ep!r!ti"n t9e p"=!rit; "# t9e 4t!ti"n!r; p9!4e i4 m!t-9e1 r"89=; ?it9 t9!t "# t9e !n!=;te4B ! m"3i=e p9!4e "# -"n4i1er!3=;
1i##erent p"=!rit; i4 t9en 4e1 #"r e=ti"n@ T9i4 pr"-e1re i4 8ener!==; m"re 4--e44#= t9!n "ne in ?9i-9 t9e p"=!ritie4 "# t9e 4"=te !n1 m"3i=e p9!4e !re m!t-9e1 3t 1i##erent #r"m t9!t "# t9e 4t!ti"n!r; p9!4e@ Here t9e 4t!ti"n!r; p9!4e "#ten -!nn"t -"mpete 4--e44#==; #"r t9e 4!mp=e -"mp"nent4B retenti"n time 3e-"me4 t"" 49"rt #"r pr!-ti-!= !pp=i-!ti"n@ At t9e "t9er extreme "# -"r4e i4 t9e 4it!ti"n ?9ere t9e p"=!rit; "# t9e 4"=te !n1 4t!ti"n!r; p9!4e !re t"" m-9 !=ie !n1 !n1 t"t! t"t!== ==; ; 1i## 1i##er erent ent #r"m #r"m t9!t t9!t "# t9e t9e m"3i m"3i=e =e p9!4 p9!4e@ e@ Here Here t9e t9e rete retent nti" i"n n time time44 3e-"m 3e-"me4 e4 in"r1in!te=; ="n8@
Procedure
'@ T9e in4trme in4trment nt ?!
Result
E##e-t "# t9e
p9! p9!4e Ine-ti"n
(ACN:H2O) +$:+$ $:,$
' ' 2
Retent Retenti"n i"n time time "# pe! ' !n1 2 (min) '@$$$'@'+$ $@6$'@+$, $@6+'@$&6
/!4e /!4e pe! pe! ?i1t ?i1t9 9 "# pe! ' !n1 2 Re4"=ti"n (min) $@$6,,$@$56' $@$5&$$@$++ $@$5$@$&&
' @ & ' @' ' @' +
E##e-t "# t9e 8r!1ient e=ti"n pr"8r!m: (ACN: H2O)
C"mp"4iti"n m"3i=e (ACN:H2O)
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p9!4e ?i1t9 "# pe! ' !n1 2 (min)
$@$$min $:,$
Retenti"n time "# pe! '
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2
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(min) $@6+5'@'$,
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'@$$''@'& $@$&'$@$+25
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A
T9e retenti"n time "# -"mp"n1 in 4t!n1!r1 mixtre !t "ptimi>e1 HPLC -"n1iti"n ($%,$) "# (ACN:H2O):
Retent Retenti"n i"n time time *t!n1!r1 -"mp"n1
in (min)
C!##eine Met9;= 3en>"!te P9en!t"=e P9en!nt9erene A-et"ne
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mixtre time
(min) ine-ti"n $@6$ '@,' 2@,$2 +@$$ '@$+,
in
'4t mixtre mixtre (min) (min) 2n1 ine-ti"n $@6+ '@,& 2@,'& +@'&& '@$&6
Discussion
Drin8 t9i4 experiment ! Hi89 Per#"rm!n-e Lii1 C9r"m!t"8r!p9; (HPLC) A8i=ent G','&A eippe1 ?it9 U 1ete-t"r + mm Re
e !n1 "34er"!te -!##eine p9en!nt9erene !n1 !-et"ne ?ere ine-te1 t9e r!ti" ?!4 -9!n8e1 t" $J:&$J !n1 $J:,$J re4pe-tie1 LC -"n1iti"n4 8ettin8 #r"m t9e !3"e1 4!mp=e@ T9ere#"re e!-9 pe! 49"=1 3e t"t!==; re4"=
T9ere !re 4"me #!-t"r4 t9!t -"ntri3te t" !== t9e pr"3=em4 4t!te1 !3"
T" i1enti#; t9e -"mp"nent4 in t9e mixtre 3; -"mp!rin8 t9e pe!4 in mixtre ?it9 t9e pe!4 "# 4t!n1!r1 -"mp"n1 t9e !=iti"!te p9en!t"=e !n1 p9en!nt9rene@
Conclusion
T9e "ptimi>e1 m"3i=e p9!4e -"mp"4iti"n #"r t9e 4ep!r!ti"n "# t9e mixtre i4 $:,$(ACN: H2O)@ T9e 9i89er -"mp"4iti"n "# "r8!ni- 4"="!te 3en>"!te next i4 p9en!t"=e pe! !n1 =!4t=; i4 -"rre4p"n14 t" p9en!nt9rene pe!@
Reference
'@ Hi89 Per#"rm!nPer#"rm!n-ee Lii1 C9r"m!t"8 C9r"m!t"8r!p9; r!p9; Lii1 Lii1 C9r"m!t"8r!p9; C9r"m!t"8r!p9;@@ Lin1e Gr"p@ Gr"p@ 2$'@ Retrie
