Lab Report 3 - Experiment Using Hplc To Separate A Pharmaceutical Mixture PDF

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HPLC lab report...

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Abs t r ac t I nt h i se xp e r i me n t ,wewi l lu s eh i g hp e r f or ma n c el i q ui dc h r o ma t o gr a ph y( HPLC)t o s e p a r a t eaph a r ma c e u t i c a lmi xt u r e .Us i n ga mi t r i p t y l i n e ,me t ha p y r i l e n e ,p h e n i r a mi n e , p r o me t h a z i n ea n dt r i pr o l i d i n e ,he x a n o p he n o n,a n dt op r e pa r et hes a mp l e .Kn o wi n gh o wt o p r e p a r et h es a mp l et op e r f o r mi nHPLC.Un d e r s t a n dh o wHPLCwo r k . Kn o wi n gh o wt or e a d c h r o ma t o gr a ph yg r a ph .Us i n gc a l i b r a t i ont ofin dwh i c hs o l u t ep a i rwi t hwh a tpe a k . De t e r mi ne i d e n t i t ywh a ti st h eun kno wn .

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I nt r o duc t i o n: Hi g hpe r f o r ma n c el i q ui dc h r o ma t o gr a p h y( HPLC)ma k e su s eofah i g hpr e s s u r ep u mpt o d e l i v e ramo bi l ep h a s es ol v e nta tau n i f o r mr a t ea tp r e s s u r e st h a ta r et y p i c a l l yf r o m5 0 0t o5 0 0 0 p s i . Th emo s to b v i o u sa d v a n t a g eo fHPLCo v e rg r a v i t yl i q u i dc h r o ma t o g r a ph yi st ha ts a mp l e sc a n b es e p a r a t e dmu c hmo r eq u i c k l y . I na d d i t i on ,s a mp l e st h a ta r en o tv o l a t i l eo rt h a two ul dt h e r ma l l y d e c o mpo s ei ng a sc h r o ma t o gr a p h yc a nb er a p i d l ya n dr o u t i n e l ys e p a r a t e di nHPLC. Co ns e q u e n t l y , t h i sp o we r f ula n a l y t i c a lme t h o di sag r e a tc o mp l e me n t a r yi n s t r u me n tt og a s c h r o ma t o gr a ph y . Ab s o r pt i onc h r o ma t o g r a p h y , t h es t a t i o n a r yp h a s ei sas ol i do fap o l a rn a t u r e s u c ha sp a r t i c l e so fh y d r a t e ds i l i c ao ra l umi n a . Th emo b i l ep ha s ea n dt h es o l ut e( c o mp o n e n t si n t h es a mpl e )a r ei nc o mp e t i t i o nf ora c t i v ea d s o r p t i ons i t e so nt h es t a t i o n a r yp ha s ep a r t i c l e s . Th u s , mo r es t r on g l ya d s o r be dc omp o n e n t sa r er e t a i ne dl on g e rt h a nwe a kl ya d s o r be dc o mp o n e n t s . Be c a u s emo r ep ol a rc o mp o u n d sa d s o r bo nap o l a rs u r f a c et ogr e a t e rd e g r e et ha ndol e s sp o l a r c o mp o u n d s , r e t e n t i o ni nt h ec o l u mni sr e l a t e dt os a mpl ep ol a r i t y . ( b o ok ) Th es t a t i o n a r yp ha s eu s e di nt h i se x p e r i me n ti ss i l i c ab a s e d ;s i l i c ai st h emo s tp o p u l a r s t a t i o n a r yp h a s es u p p o r tf o rLC.Si n c et heb a r es i l i c ap a r t i c l e sa r ep o l a r ,t h eno n p o l a rs t a t i o na r y p h a s er e qu i r e df o rRPLCi so b t a i n e dt h r ou g ht h eb o n di n go fo c t y l d e c y lc h a i n so n t ot h es u r f a c eo f t h es i l i c a .Th i se x pe r i me n twi l lf o c u so nt h eq u a nt i fic a t i o no fe a c hp h a r ma c e u t i c a ls ol u t ei na n u n kn o wnmi x t ur ea n dar e a lwo r l da n t i h i s t a mi n es a mpl e( Ac t i f e d ) .Kn o wi n gt h ea b u n d a n c eofa p a r t i c u l a rs o l u t ei se xt r e me l yus e f u li nt h ep h a r ma c e u t i c a li n d u s t r yt ov e r i f yt hep u r i t yofa s u b s t a n c eo rt od e t e r mi n et h ey i e l do fas y n t he s i s . Th efir s tp e a ki nac h r o ma t o g r a mc a nb eau s e f u ld i a g no s t i ct o o lf o rt r o u b l e s ho o t i n g l i qu i dc h r o ma t o gr a p h i c( LC)s e p a r a t i o ns .Mo s tpe o p l er e f e rt ot h i sa st h ec o l u mnd e a dt i mep e a k , 2

a b br e v i a t e dt0 . Ho we v e r , i tha sawi d ev a r i e t yo fo t he rn a me s :j u n kpe a k ,g a r b a g ep e a k ,s o l v e n t f r o n t , orh o l d u pt i me , wi t htM a st h emo s tc ommo na l t e r na t i v ea b b r e vi a t i o n. Th i sr e p r e s e n t st h e t i mei tt a k e ss o me t h i n gt og ot h r o ug ht h eLCc o l umnt h a td o e sn o ti n t e r a c twi t ht h ec o l u mn .A c o r r e s po n d i n gd e a dv o l u me( o rh o l d u pv o l ume ) , VM, i st h ev ol u meo fmob i l ep h a s ei n s i d et he c o l u mn .Th i sv o l u mec o mp r i s e sb o t ht h ev o l u meo fmobi l ep h a s eb e t we e nt h ep a c ki n gp a r t i c l e s ( t h ei nt e r s t i t i a lv o l ume )a n dt h ev ol umewi t hi nt h ep a r t i c l e s( t h ep o r ev o l u me ) . We ' l ls e et h a tt0 c a nb eau s e f u ld i a g n o s t i ct oo lt oi d e n t i f ypo t e n t i a lp r ob l e mswi t ha nLCme t h o d .( * ) Re s o l u t i o n Th emo s tc o mmo ng o a li nRPLCi st oob t a i na d e qu a t es e pa r a t i o no fe a c hs o l u t ei na r e a s o na b l ea n a l y s i st i me ;a de q u a t es e p a r a t i o na l l o wso n et oi s o l a t eas pe c i ficc o mp o n e n tf r o ma mi x t u r ef o rt h ep ur p o s e so fi d e nt i fic a t i o na n d/o rq u a n t i fic a t i o n .Re s o l u t i o ni same a s u r eo ft h e c o mp l e t e n e s sofas e p a r a t i o na n di sq u a n t i t a t i v e l yme a s u r e dwi t ht h eg e n e r a le q u a t i o n :

Wh e r et R, 1a n dt R, 2r e pr e s e n tt h er e t e n t i ont i me soft h el e a s ta n dmo s tr e t a i n e dp e a k s , r e s p e c t i v e l y , me a s u r e da tt h ep e a kc e n t r o i d( i . e . , c e n t e ro fma s s )a n dwb , 1a n dwb , 2r e pr e s e n tt he b a s e l i n ewi d t h so ft h ep e a ks( e q u a lt of o urt i me st h es t a n d a r dd e v i a t i o noft h ep e a k , a s s u mi n ga Ga u s s i a np e a ks h a p e ) .Ba s e l i n e( i . e . , c o mp l e t e )s e p a r a t i ono ft wop e a ksi so b t a i n e dwh e nRs3 1 . 5.Th er e s o l ut i onh a sn ou n i t s ;t h u s , t h er e t e n t i o nt i mea n dp e a kwi d t hmus tb eme a s u r e di nt h e s a meun i t s( i . e . ,t i me , v o l u meo rd i s t a n c e ) .Re f e rt oFi g u r e2f o ra ni l l u s t r a t i o nofs e p a r a t i o n

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b a s e do nv a r i o u sv a l u e so fr e s o l u t i o n. Rsi sc a l c ul a t e du s i n ge q. 1. Atl o wRs , p e a k sa r eb a r e l y s e p a r a t e da ndRs30 . 7i sr e q u i r e dt od i s t i n g ui s hbe t we e nt wop e a k s . Co l u mnEffic i e n c y An ot h e rwa yt ome a s u r et h es e p a r a t i n gp o we ro fac ol u mni st ome a s u r et h ec o l u mn e ffic i e n c y( N) , wh i c hi st y pi c a l l yr e f e r r e dt oa st hen u mb e ro ft h e o r e t i c a lpl a t e s . Co l u mn e ffic i e n c yi sme a s u r e dwi t ht h ef o l l o wi n ge q ua t i o n :

wh e r et Ri st h er e t e n t i o nt i mea tt hepe a kc e nt r o i da n dw1 / 2i st h eh a l f wi d t hoft h epe a k ( i . e . , p e a kwi dt hme a s u r e da tt h eh a l f h e i g htoft hep e a k ) . W1 / 2v a l u e sa r et h eme a s u r e me n t s a c t u a l l yb e i n gr e p o r t e di ny o u rc hr o ma t o g r a ms . Bo t ho ft he s ev a l u e sa r eob t a i n e dd i r e c t l yf r o m t h ed a t as y s t e mi nt h i se x p e r i me n t . Pe a kCa p a c i t y Ac o mmonc o n c e r ni nc h r o ma t o gr a p h yc e n t e r sa r o u n dt h ec o n c e pto fp e a kc a p a c i t y( n c ) , whi c hi sd e fin e da st h en u mbe ro fp e a k st h a tc a nfil lag i v e ns e p a r a t i o ns p a c ea s s u mi n gt h a tt he p e a k sa r eo pt i ma l l ys p a c e d . On ei mp o r t a n to b s e r v a t i o nwh e nd i s c u s s i n gp e a kc a p a c i t yi st ha t e a c ho b s e r v e dp e a ki sn o tn e c e s s a r i l yas i n g l ec o mpo ne n t .Al s o, ac o mp o ne n ti nt h es a mp l ei sno t r e s t r i c t e dt oe l u t i n gf r o mt h ec o l umnwi t h i nt h ec ho s e ns e p a r a t i o ns pa c e .Th e r e f o r e ,t h e p r o b a b i l i t yo fo b s e r vi n gs i n g l ec o mp on e n tp e a k si na n ys e pa r a t i o ni so f t e nv e r yl o w( l e s st ha n 2 0 %)( 2 ) , wh i c hi swh yt h ec h o i c eo ft h ea p pr o p r i a t ec h r oma t o gr a p h i cc o nd i t i o ns( i . e . ,s t a t i on a r y

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p h a s e , mob i l ep h a s e , e l u e n tc o mp o s i t i on , t e mp e r a t u r e , e l u t i o nmo d e , e t c . )i sp e r t i ne n tt o o b t a i n i n gc o mp l e t es e pa r a t i o no fe v e r yc o mp on e nt . I ni s o c r a t i ce l u t i o n , t hep e a kc a p a c i t yi sd e t e r mi ne dwi t ht h ef o l l o wi n ge q ua t i o n :

wh e r ek ’ nr e p r e s e nt st h er e t e n t i onf a c t o r( k ’ )o ft h el a s te l u t i n gc o mp o n e n t( n ) . Th e r e t e n t i o nf a c t o ri sc a l c u l a t e du s i n gt h ef o l l o wi n ge q u a t i o n :

wh e r et oi st h ed e a dt i meo ft h ec o l u mn( weme a s u r et h i sv a l ueus i n gt heu n r e t a i n e d s o l ut eo fu r a c i l ) .I ng r a d i e n te l u t i o n , t hepe a kc a pa c i t yi sd e t e r mi n e dwi t has i mi l a re q u a t i o na si n i s oc r a t i ce l u t i on

As s u mi n gt h a tt hep e a kwi d t hi sc o n s t a n ti ng r a d i e nte l u t i o n. Ba s e dont h ef o r mo f e q ua t i o n s3a n d5 ,i ts h o u l db ec l e a rt h a t , t h e o r e t i c a l l y , gr a d i e n te l ut i o nwi l la l wa y sp r o vi d ea h i gh e rp e a kc a p a c i t yc o mp a r e dt oi s o c r a t i ce l u t i o n , a s s u mi n gt h a tt h ev a l u eo fk ’ ni sc o mp a r a bl e i ne a c he l u t i o nmo d e .

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Ex pe r i me nt a l Eq u i p me n t : - Th emi c r o p i pe t t e sl a bki t :1 0 . 0 0mLv o l u me t r i cfla s ks , t e s tt u b e swi t hc o r ks t o p p e r s , di s p o s a b l e1mLp l a s t i cs y r i n g e swi t h0 . 2 2μmn y l o nfil t e rt i ps ,a n da nOTCa n t i h i s t a mi n e s ol u t i o n .2mi c r o p i p e t t e s :1 0–1 0 0μLEp pe n d o r fp i p e t t e ,a n d1 0 0–1 0 0 0μLEppe n d o r f pi p e t t e - Ami t r i p t y l i n e( 1 0mg / mL) , Me t h a p y r i l e n e( 1 0mg / mL), Ph e ni r a mi n e( 1 5mg / mL) , Pr o me t ha z i n e( 1 0mg / mL) , Tr i p r o l i d i n e( 1 0mg / mL) , He xa n o p h e n o n e( I n t e r na lSt a n d a r d ) ( 5mg / mL) ,Ur a c i l( De a dTi meMa r k e r )( 1mg / mL) - Un kn o wnF Pr e p ar ek n o wnmi x t u r es o l u t i o n: - 5o f1 0 . 0 0mLv o l u me t r i cfla s k swe r el a b e l e df r o m 1t o5 - 10–1 0 0μLEp p e n d o r fpi p e t t ewa su s e dt ome a s u r eou tkn o wns o l u t ef o l l o wt h i st a b l e : Ta bl ev o l umea dde d

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- di l ut i n gt h e s e5fla s ku s e dEl u e n tAb yfil l i n gt h efla s kwi t hEl u e ntAu n t i li tr e a c ht h e da s hl i ne . - Th efla s kwa sc o v e r e db yt h es t o pp e ra n di n v e r ti tg e n t l ya tl e a s t6t i me ss oi tc a nb e t h o r o u g hl ymi x .(doi tt oe a c ha nde v e r yfla s kf r o m 1t o5 ) Pr e p ar eu nk n ownmi x t u r es o l u t i o n - l a b e l e da n o t h e r1 0. 0 0mLv o l u me t r i cfla s ksu n kn o wn - di l ut e1 / 2 0u n kn o wns a mpl ei nEl u e n tAu s e d1 0 . 0 0mLfla s kwh i c hme a n1 / 2 0o f1 0ml 1 x 10 mL =0.5 ml 20 0 . 5 mL=5 0 0 μL ( 1 mL=1 0 00μL)

- 10 0–1 0 0 0μLEp p e n d or fp i p e t t ewa su s e dt ome a s u r eo u t5 00μLofu nkn o wna n d de l i v e r e dt o1 0 . 0 0mLv o l u me t r i cfla s ksl a b e l e du n kn o wn . - Us e d1 0–1 0 0μLEp p e n d o r fp i p e t t et ome a s u r e5 0μLo fUr a c i l( De a dTi meMa r ke r )( * ) a n d9 0μL, He x a n o p h e n o n e( I nt e r na lSt a n d a r d )( 5mg / mL) - Fi l l e dt h efla s kwi t hEl u e ntAt ot h ed a s hl i n e . - Th efla s kwa sc o v e r e db yt h es t o pp e ra n di n v e r ti tg e n t l ya tl e a s t6t i me ss oi tc a nb e t h o r o u g hl ymi x . Pr e p ar eHPLCs a mpl e s : - 5Ep p e n d o r ft u b e swe r el a b e l e df r o m 1t o5 , 1Ep p e n d o r ft u b e sl a b e l e du n kn o wnF - e a c hs a mp l ewi l lbefil t e r e dt ot h os el a b e l e d1 mLEp p e n d o r ft u b e . - Fi l l e d1mLp l a s t i cs y r i n g e swi t ht hes a mp l ef r om t h efla s k . Fi l l e dt oe x a c t1mLt h e np ut t h en y l o nt i pfil t e ro nt hes yr i n g ea n dp us h e dt h es o l u t i o nt hr o u g ht h efil t e ri nt ot h e ma t c h i n gt u be . 7

- On eBLANKs a mp l emu s tb ep r e p a r e da l s o .Us i n gt h es a met e c h n i q u el i s t e dt ofil lt h e pl a s t i cs y r i n g e swi t hj u s tEl u e n tA,a ndpu s h e dt h es o l u t i o nt h r o u g ht hefil t e ri n t oa n o t h e r t u b el a b e l e dBLANK. Dat ac o l l e c t e df r o mHPLC: Th eo r d e ro fr u n n i n gt h e s es a mp l ewi l lbeb l a n ks a mp l efir s tt ot e s to u tt hei n s t r u me n tl a s t t i meb e f o r er unt h er e a ls a mp l e . Ne xt , wi l lb er u nt h e5mi x t u r es a mp l e se a c ho ft h e mo n c e ,a n d l a s t l yr u nt h eu n kn o wnt hr e et i me s . 50μLs y r i n g ewa su s e dt oi n j e c tt h es a mp l et oi n s t r u me n t .Be f o r ed ot h er e a li n j e c t i o n r i n s e st h es yr i n g et h r e et i me swi t ht h es a mp l ei twi l lu s et oi n j e c tt oe l i mi n a t eu n wa nt e ds o l u t e . Re s ul t s : Fr o mt h eHPLCr e po r tt h e r ea r ema n l y7p e a k s( l a r g ee n o u g ht on o t i c e )t h efir s tp e a kwi l lb e Ur a c i l( De a dTi meMa r k e rr e a d( * )i ni n t r o d u c t i o n ),t h el a s tp e a kwi l lbeHe x a n o p h e n o n e ( I n t e r n a lSt a n da r d ) . Th e r ea r e5p e a k sl e f ti nt h emi d d l e , t h e s e5p e a kswe r el a b e l e df r o m 1t o5 f r o ml e f tt or i g h t . I tma k e st hed a t ae a s yt os e ea n dc o l l e c t . Ta bl e1 :Ar e aunde rt hepe a k

Solution 1 2 3 4 5 unknown F1 unknown F2 unknown F3

Hexanophenoe Area % under the peak (AH) 0.4526 0.5756 0.4025 1.5216 0.5025 0.8873 2.0014 0.8658

Area % under the peak 1(A1) 0.5451 0.8724 0.2523 0.9365 0.5576 0.5564 1.3287 0.5965

Area % under the peak 2 (A2) 2.9605 2.2512 1.7958 7.1729 3.5140 1.7680 4.2000 1.8918

Area % under the peak 3 (A3) 1.9221 2.9394 2.3743 9.2844 2.0889 1.7704 4.2042 1.875

Area % under the peak 4 (A4) 1.6089 2.8996 2.1332 3.4018 1.8628 1.228 2.9703 1.2807

Area % under the peak 5 (A5) 1.1334 2.1488 1.5822 6.7149 3.0908 1.6974 3.9877 1.7233

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Fr o mt h ed a t at a b l e1c r e a t e dat a b l es h o wt h er a t i ob e t we e nt hea r e au n d e rt h ep e a kv st h ea r e a u n d e rHe x a n o p h e n o e , b e c a u s eHe x a n o p h e n o ei si n t e r n a ls t a n d a r dv a l u ewh i c hwo n ’ tc h a n g e t h ou g h to u tt h i se x p e r i me nt .

Ta bl e2 :Pe a kar e ar a t i o( s o l ut eo v e rHe x a no phe no ne ) Solution 1 2 3 4 5 unknown F1 unknown F2 unknown F3

(A1) /(AH) 1.20437472 4 1.51563585 8 0.62683229 8 0.61547055 7 1.10965174 1 0.62707088 9 0.66388528 0.68895818 9

(A2)

/(AH)

6.54109589

(A3) /(AH) 4.24679628 8

(A4) /(AH) 3.55479452 1

3.91104934 4.46161490 7 4.71405099 9 6.99303482 6 1.99256170 4 2.09853102 8 2.18503118 5

5.1066713 5.89888198 8 6.10173501 6 4.15701492 5 1.99526653 9 2.10062955 9 2.16562716 6

5.03752606 5.29987577 6 2.23567297 6 3.70706467 7 1.38397385 3 1.48411112 2 1.47920997 9

(A5) /(AH) 2.50419796 7 3.73314801 9 3.93093167 7 4.41305205 6.15084577 1 1.91299447 8 1.99245528 1 1.99041349

Co nc e n t r a t i o no ft h eg i v e ns o l u t ec a nb ef ou n du s i n gt h i s C1V1=C2V2

C2=( C1V1) /V2

Wh e r eC1g i v e n( i ne x p e r i me n tu n d e re q u i p me n ts e c o n dd a s h)V1a l s og i v e n( Ta b l ev o l ume a d de d )V2 wi l lb e1 0mLa ta l lt i meb e c a u s et h et o t a lv o l u mea tt hee n dwa s1 0mL.Fr om h e r e p u ta l lda t ai n t ot hi st a b l e . Ta bl e3 :c o nc e nt r a t i o no fs o l ut ei ne a c hs ol ut i o n: s o l u t i o n

Con c e n t r a t i o no f Ph e ni r a mi n e ( mM)

Co nc e n t r a t i o n Co n c e n t r a t i o Co n c e n t r a t i o Co n c e n t r a t i o o f nof no f no f Me t ha p y r i l e n Tr i p r o l i d i n e Pr o me t h a z i n e Ami t r i p t y l i n e e ( mM) ( mM) ( mM) 9

1 2 3 4 5

( mM) 0.08 0.07 0.06 0.05 0.04

0.105 0.12 0.06 0.075 0.09

0.05 0.06 0.07 0.08 0.04

0.06 0.07 0.08 0.04 0.05

0.04 0.05 0.06 0.07 0.08

Fr o mt a b l e2a nd3wec a na s s u mepe a k5p r e s e n t i n gAmi t r i p t y l i n ebe c a u s et h eAmi t r i p t y l i n e c o nc e n t r a t i o ni n c r e a s ea n dpe a k5b e h a v es a mewa y . Pe a k4i sp r e s e n t i n gPr o me t h a z i n eb e c a u s e t h efir s t3t r a i li ti n c r e a s e dt h a nd e c r e a s e da n dg ou pa g a i n . Pe a k3i sp r e s e n t i n gTr i p r o l i di n e b e c a u s efir s t4p e a k si n c r e a s e da n dl a s to n ei sd r o pd o wne x a c th o wt h ec o n c e n t r a t i o nb oa r d b e ha v e . Pe a k2a n dp e a k1a r eo v e rp l a c es oi ti sha r dt og e tab e s ta s s u mp t i o n . Ho we v e r ,p e a k1 b e ha v ec l o s e l ys a mea sPh e n i r a mi n efir s tt wope a k si n c r e a s e da n dl a s t3d e c r e a s e ds ot h el a s ton e p e a k2wi l lb eMe t h a p yr i l e n e . Me t h a p y r i l e n er a t i or e a l l yh a r dt os e ei tb e ha v es ot h eb e s twa yt o d oi sus i n ge l i mi n a t i o n .Us ei n f o r ma t i o nf r om t a bl e2a n d3 , c r e a t e d5g r a p h sma t c h i n gt h er a t i o

Area of Pheniramine over area of Hexanophenone

a n de a c hc o n c e n t r a t i on :

Pheniramine Gr a ph1 :Phe ni r ami ne

0.14 0.12

c o nc e nt r a t i ona nd

f(x) = 0.06 x + 0.03 R² = 0.92

0.1 0.08

a r e aunde rt hepe a k

0.06

r a t i o

0.04 0.02 0 0.4

0.6

0.8

1

1.2

1.4

1.6

Pheniramine concentration

10

Area of Methapyrilene over area of Hexanophenon Area of Triprolidine over area of Hexanophenone

Methapyrilene Gr a ph2 :

8 7

Me t ha py r i l e ne

6 f(x) = − 17.07 x + 6.35 R² = 0.04

5 4

c o nc e nt r a t i o na ndar e a

3 2

unde rt hepe a kr a t i o

1 0 0.04

0.04

0.05

0.05

0.06

0.06

0.07

0.07

0.08

0.08

0.09

Methapyrilene concentration

Triprolidine Gr a ph3 :Tr i pr ol i di ne

7 6

f(x) = 55.42 x + 1.78 R² = 0.94

5

c o nc e nt r a t i o na nda r e a unde rt hepe a kr a t i o

4 3 2 1 0 0.04

0.04

0.05

0.05

0.06

0.06

0.07

0.07

0.08

0.08

0.09

Triprolidine concentration

Gr a ph4 :Pr ome t ha z i ne

Area of Promethazine over area of Hexanophenone

Promethazine

c o nc e nt r a t i o na nda r e a

6 5 4

f(x) = 74.59 x − 0.51 R² = 0.9

unde rt hepe a kr a t i o

3 2 1 0 0.04

0.04

0.05

0.05

0.06

0.06

0.07

0.07

Promethazine concentration (M)

0.08

0.08

0.09

11

Area of Amitriptyline over area of Hexanophenone

Amitriptyline Gr a ph5 :Ami t r i pt y l i ne

7 6 5

c o nc e nt r a t i o na nda r e a

f(x) = 79.73 x − 0.64 R² = 0.91

4

unde rt hepe akr a t i o

3 2 1 0 0.04

0.04

0.05

0.05

0.06

0.06

0.07

0.07

0.08

0.08

0.09

Amitriptyline concentration

Fr o mt h eg r a p ha bo v e ,t h e r ea r e5l i ne a re q u a t i o n sb e s tfitf o re a c hg r a p h . y=mx+b yi sr a t i op e a ka r e a xi st h ec o n c e n t r a t i on Fr o m Ta b l e2:Pe a ka r e ar a t i ot h eyv a l u e sa r eg i v e n ,o n l yn e e dt ofin dxv a l u e . So l v et hee q ua t i o n f o rxwi l lg i v et hec o n c e n t r a t i o ni nt h eu n kn o wn ....