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7/26/2019 6.Results and Discussion
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RESULTS AND DISCUSSION
Method Development
Trail 1:
Column : Symmetry C18 (4.6250mm)5
Column temperature : Ambient
Waelen!t" : 262 nm
#obile p"a$e ratio : #et"anol (100%)&'&
lo rate : 1ml'min
*n+e,tion olume : 10l
-un time : 8min
i!ure: ,"romato!ram or trail 1
Table: peak re!lt "or trail 1
S#No $eak Name R t Area %ei&htUS$
Tailin&
US$ $late
'o!nt
1 /ramaol1.50 1243 02
2.5 16
Obervation:
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*n t"i$ trial it $"o$ le$$ plate ,ount an more tailin! an improper $eparation o pea in t"e
,"romato!ram. So it reuire$ more trial$ to obtain !oo pea$.
Trail (:
Column : terra C18 (4.6250mm)5
Column temperature : 407C
Waelen!t" : 262 nm
#obile p"a$e ratio : #et"anol:Water (65:35 %)&'&
lo rate : 1ml'min
*n+e,tion olume : 10l
-un time : 10min
i!ure: ,"romato!ram or trail 2
Table: peak re!lt "or trail (
S#
No$eak name R t Area %ei&ht
US$
Tailin&
US$ plate
'o!nt
1 /ramaol 2.104 12668 6 2. 634
Obervation: *n t"i$ trial it $"o$ le$$ plate ,ount more plate ,ount an improper $eparation
o pea in t"e ,"romato!ram. So it reuire$ more trial$ to obtain !oo pea$.
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Trail ):
Column : *nert$il C18 (4.6250mm)5
Column temperature : 409C
Waelen!t" : 262nm
#obile p"a$e ratio : AC:Water: (20:80 %)&'&
lo rate : 1ml'min
*n+e,tion olume : 10l
-un time : 5min
i!ure; ,"romato!ram or trail 3
Table: * peak re!lt "or trail )
S# No $eak name R t Area %ei&htUS$
Tailin&
US$ plate
'o!nt
1 /ramaol 2.668 11542 635 2.14 335
Obervation: *n t"i$ trial it $"o$ le$$ plate ,ount more tailin! ee,t an improper
$eparation o pea in t"e ,"romato!ram. So it reuire$ more trial$ to obtain !oo pea$.
Trail +:
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Column :
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Column : ;/erra ->18 (4.6250mm) 5
Column temperature : 309C
Waelen!t" : 262nm
lo rate : 1ml'min
*n+e,tion olume : 10l
-un time : 5min
i!ure:
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i!ure:
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i&: Chromato&ram ho2in& blank -mobile phae preparation.
S3tem !itabilit3:
i&: Chromato&ram ho2in& in4e'tion *1
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i&: Chromato&ram ho2in& in4e'tion *(
i&: Chromato&ram ho2in& in4e'tion *)
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i&: Chromato&ram ho2in& in4e'tion *+
i&: Chromato&ram ho2in& in4e'tion *5
/able: -e$ult$ o $y$tem $uitability or /ramaol
S#No $eak Name RT
Area
-6/7e'.
%ei&ht
-6/. US$ $late Co!nt US$ Tailin&
1 /ramaol 2.13 505628 415 581 1.4
2 /ramaol 2.165 50584 454466 5810 1.4
3 /ramaol 2.12 506855 433328 5 1.60
4 /ramaol 2.15252066
430583 55 1.53
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5 /ramaol 2.128 501 4421 583 1.54
Mean 50648.6
Std# Dev# 6318.103
8 RSD 1.2
A''eptan'e 'riteria:
%-S= o ie ierent $ample $olution$ $"oul not more t"an 2
/"e %-S= obtaine i$ it"in t"e limit "en,e t"e met"o i$ $uitable.
S$ECIICIT9
/"e *C@ o,ument$ eine $pe,ii,ity a$ t"e ability to a$$e$$ uneuio,ally t"e analyte in t"e
pre$en,e o ,omponent$ t"at may be epe,te to be pre$ent $u," a$ impuritie$ e!raation
prou,t$ an matri ,omponent$.
Analyti,al met"o a$ te$te or $pe,ii,ity to mea$ure a,,urately uantitate /ramaol in
ru! prou,t.
Aa3 -Standard. :
i!. C"romato!ram $"oin! a$$ay o $tanar in+e,tion ;1
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i!. C"romato!ram $"oin! a$$ay o $tanar in+e,tion ;2
i!. C"romato!ram $"oin! a$$ay o $tanar in+e,tion ;3
Table: $eak re!lt "or aa3 tandard
S#No Name RT Area %ei&ht US$Tailin& U S$$late
Co!nt
In4e'tion
1 /ramaol 2.180 522 482601 1.44 585 1
2 /ramaol 2.168 55 4316 1.42 5814 2
3 /ramaol 2.16 564 48126 1.50 584 3
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Aa3 -Sample.:
i&: Chromato&ram ho2in& aa3 o" ample in4e'tion*1
i Chromato&ram ho2in& aa3 o" ample in4e'tion*(
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i&: Chromato&ram ho2in& aa3 o" ample in4e'tion*)
/able: >ea re$ult$ or A$$ay $ample
S#No Name RT Area %ei&ht US$Tailin& US$$late
Co!nt
In4e'tion
1 /ramaol 2.1 5661 46388 1.4 5820 1
2 /ramaol 2.184 53525 4600 1.50 5810 2
3 /ramaol 2.186 54005 463262 1.52 5841 3
%ASSAB
Sample area Wei!"t o $tanar =ilution o $ample >urity Wei!"t o tablet
DDDDDDDDDDD DDDDDDDDDDDDDDDD DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD100
Stanar area =ilution o $tanar Wei!"t o $ample 100 Eabel ,laim
55431'566210'3030'0.0182.'1000.182'100100
100.8%
/"e % purity o /ramaol in p"arma,euti,al o$a!e orm a$ oun to be 100.8%%.
LINEARIT9
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i Chromato&ram ho2in& linearit3 level*1
i Chromato&ram ho2in& linearit3 level*(
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i Chromato&ram ho2in& linearit3 level*)
i Chromato&ram ho2in& linearit3 level*+
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i Chromato&ram ho2in& linearit3 level*5
C%ROMATORA$%IC DATA OR LINEARIT9 STUD9:
Con'entration
Level -8.
Con'entration
&;ml
Avera&e
$eak Area
33 20 168581
66 40 3540
100 60 52258
133 80 66532
166 100 8556
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LINEARIT9 $LOT:
/"e plot o Con,entration () er$u$ t"e Aera!e >ea Area (y) ata o /ramaol i$ a $trai!"t
line.
B m F ,
Slope (m) 8628
*nter,ept (,) 130
Correlation Coei,ient (r) 0.
/ALIDATION CRITERIA:/"e re$pon$e linearity i$ eriie i t"e Correlation Coei,ient i$
0. or !reater.
CONCLUSION: Correlation Coei,ient (r) i$ 0. an t"e inter,ept i$ 130. /"e$e alue$
meet t"e aliation ,riteria.
$re'iion:
/"e pre,i$ion o an analyti,al pro,eure epre$$e$ t"e ,lo$ene$$ o a!reement (e!ree o
$,atter) beteen a $erie$ o mea$urement$ obtaine rom multiple $amplin! o t"e $ame
"omo!eneou$ $ample uner t"e pre$,ribe ,onition$.
RE$EATA0ILIT9
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i Chromato&ram ho2in& pre'iion in4e'tion *)
i Chromato&ram ho2in& pre'iion in4e'tion *+
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i Chromato&ram ho2in& pre'iion in4e'tion *5
Table: Re!lt o" repeatabilit3 "or Tramadol:
S# No $eak nameRetention
timeArea-6/7e'.
%ei&ht
-6/.
US$ $late
Co!nt
US$
Tailin&
1 /ramaol 2.18 50041 446535806 1.55
2 /ramaol 2.14 50453 444485803 1.60
3 /ramaol 2.10 512545 440821585 1.60
4 /ramaol 2.1 505013 44658 1.56
5 /ramaol 2.12 506855 4333285 1.60
Mean 5018.2
Std#dev 3185.43
8RSD 0.62
A''eptan'e 'riteria:
%-S= or $ample $"oul be #/ 2
/"e %-S= or t"e $tanar $olution i$ belo 1 "i," i$ it"in t"e limit$ "en,e
met"o i$ pre,i$e.
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Intermediate pre'iion:
Anal3t 1:
i&: Chromato&ram ho2in& Anal3t 1 in4e'tion *1
i&: Chromato&ram ho2in& Anal3t 1 in4e'tion *(
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i&: Chromato&ram ho2in& Anal3t 1 in4e'tion *)
i&: Chromato&ram ho2in& Anal3t 1 in4e'tion *+
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i&: Chromato&ram ho2in& Anal3t 1 in4e'tion *5
i&: Chromato&ram ho2in& Anal3t 1 in4e'tion *3.345.'8628
2.86!'ml
!antitation limit
/"e uantitation limit o an iniiual analyti,al pro,eure i$ t"e loe$t amount o
analyte in a $ample "i," ,an be uantitatiely etermine.
LO>1=?@;S
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W"ere
G Stanar eiation o t"e re$pon$e
S Slope o t"e ,alibration ,ure
Re!lt: >1045.'8628
8.6!'ml
Rob!tne
/"e robu$tne$$ a$ perorme or t"e lo rate ariation$ rom 0. ml'min to 1.1ml'min an
mobile p"a$e ratio ariation rom more or!ani, p"a$e to le$$ or!ani, p"a$e ratio or
/ramaol. /"e met"o i$ robu$t only in le$$ lo ,onition an t"e met"o i$ robu$t een by
,"an!e in t"e #obile p"a$e H5%. /"e $tanar an $ample$ o /ramaol ere in+e,te by
,"an!in! t"e ,onition$ o ,"romato!rap"y. /"ere a$ no $i!nii,ant ,"an!e in t"e
parameter$ lie re$olution tailin! a,tor a$ymmetri, a,tor an plate ,ount.
/ariation in "lo2
i&!re: 'hromato&ram ho2in& le "lo2 o" =#Bml;min
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i&!re: 'hromato&ram ho2in& more "lo2 o" 1#1 ml;min
/ariation o" mobile phae or&ani' 'ompoition
i&!re: 'hromato&ram ho2in& le or&ani' 'ompoition
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i&!re: 'hromato&ram ho2in& more or&ani' 'ompoition
Table: Re!lt "or Rob!tne
$arameter !ed "or ample anal3i $eak Area Retention Time Theoreti'al
plateTailin& "a'tor
A,tual lo rate o 1.0 mE'min 56012 2.165 5846 1.41
Ee$$ lo rate o 0. mE'min 54838 2.8 5626 1.28
#ore lo rate o 1.1 mE'min 4855 1.2 532 1.33
#ore