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PRINSIP DASARHPLC

BAGIAN I YUNIKA MAYANGSARI, S.Si., M.Biotech

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Fasa diam dan asa !e"a# $HPLC%

Da&am HPLC, 'at cai" $&i()id% di!)na#anse*a!ai asa !e"a#

Ko&om *e"isi se"*)# ha&)s +an! diadat#an$se*a!ai asa diam%

 -he stationa"+ hase is dened as theimmo*i&e ac#in! mate"ia& in the co&)mn.

Daat di*a+an!#an *etaa s)&itn+a 'at cai"men!a&i" me&a&)i asa diam di da&am #o&om

Sehin!!a, a!a" 'at cai" daat me&e/ati#o&om den!an ceat, di*)t)h#an *ant)anoma *e"te#anan tin!!i

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Pe"samaan den!an GC  #e&)a"ann+a *e")a#"omato!"am

Ke)nt)n!an di*andin!ema#aian GC  #emam)anmen!ana&isis same& +!

unvolatile dan &a*i& ada s)h)tin!!i

A#an tetai ana&isis HPLC &e*ih

maha&

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Advantages to HPLC

Hi!he" "eso&)tion and seed o ana&+sisHPLC co&)mn can *e "e)sed /itho)t

"eac#in! o" "e!ene"ation

G"eate" "e"od)ci*i&it+ d)e to c&osecont"o& o the a"amete"s a1ectin! thee1ecienc+ sea"ation

2as+ a)tomation o the inst")ment

oe"ation and data ana+sisAdata*i&it+ to &a"!e3sca&e, "ea"ati4e

"oced)"s

*asic "inci&es o HPLC 3 5s)/edo hadi/i+oto 6

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7a"io)s inst")ments o HPLC s+stem

*asic "inci&es o HPLC 3 5s)/edo hadi/i+oto 8

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S#ema Inst")men HPLC

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P"insi #e":a HPLC

Den!an *ant)an oma ase!e"a# dia&i"#an me&a&)i #o&om #edete#to". Same& +an! di&a")t#an

da&am so&4ent, dimas)##an #eda&am a&i"an asa !e"a# den!anca"a in:e#si. Di da&am #o&om

te":adi emisahan #omonen;cam)"an  e"*edaan #e#)ataninte"a#si anata"a ana&at $so&)t3so&)t% den!an stationary   phase ada #o&om.

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P"insi #e":a HPLC $&an:%

So&)t3so&)t +an! #)"an! #)at inte"a#sin+aden!an ase diam a#an #e&)a" da"i #o&om

te"&e*ih dah)&). Se*a&i#n+a, so&)t; +an! #)at*e"inte"a#si den!an asa diam ma#a so&)t; ts*a#an #e)a" da"i #o&om &e*ih &ama. Setia#omonen cam)"an +an! #e&)a" da"i #o&om

didete#si o&eh dete#to" #em)dian di"e#amda&am *ent)# #"omato!"am.

K"omato!"am HPLC se")a den!an#"omato!"am GC   :)m&ah ea# men+ata#an :)m&ah #omonen< &)as a"ea ea# men+ata#an

#onsent"asi da&am cam)"anSisitim HPLC daat dih)*)n!#an den!an

sot/a"e ada #om)te" dan dioe"asi#anseca"a com)te"i'e

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Inst")mentasi HPLC

Fase !e"a#Poma

Sam&e in:ecto"

Ko&om

Dete#to"

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Fasa Ge"a# $MP% HPLC $S+a"atasa !e"a#%MP ha")s *e"tinda# s*! e&a")t +an!

*ai# )t# same& +an! diana&isis

MP ha")s m)"ni se#a&i )t# men!hinda"iadan+a im)"ities +an! a#anmen!!an!!) inte"etasi ada#"omato!"am dan men!oto"i #o&om

MP ha")s :e"nih se#a&i )t#

men!hinda"#an #o&om #oto" dante"s)m*at. Pe&a")t ha")s disa"in! dandide!as. Sa"in!an di!)na#an ni&ondiamete" =.68 mi#"omete".

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MP m)dah die"o&eh, tida#m)dah te"*a#a", dan tida#

*e"ac)nMP tida# 4is#o)s. Ke#enta&an

tida# me&e*ihi =,8 cP $centiPoise%

MP ses)ai den!an dete#to"n+a.Unt)# dete#to" RI $"e"acti4einde>% e&a")t ha")s mem)n+ai

inde#s *ias +an! *e"*eda den!anso&)t< )t# det. U7, e&a")t td#*o&eh mn+e"a caha+a ada!e&om*an! caha+a +! dia#ai.

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 ?enis asa !e"a#

Be"dasa"#an #eo&a"an asa diam dan asa!e"a#

Fase No"ma& $No"ma& Phase%

Kom*inasi anta"a ase diam o&a" dan ase

!e"a# non3o&a" $misa&@ ase diam@ si&i#aata) a&)mina, ase !e"a#@ he#sana ata) i3"oi&ete"%

Fase -e"*a&i# $Re4e"sed Phase%

Fase diam non3o&a" dan ase !e"a#n+a o&a"$ai", metano&, asetonit"i&%  #eo&a"an ase!e"a# &e*ih tin!!i di*andin! ase diamn+a

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P)m

F&o/ "ate "an!e@ =.=5 to 8mLmin

F&o/ "ate sta*i&it+@ not mo"e than

5Fo" o/ "ate sta*i&it+ sho)&d *e

&ess than =.;

It is desi"a*&e to ha4e aninte!"ated de!assin! s+stem,eithe" he&i)m )"!in!, o"

mem*"ane &te"in!.

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Pump

 -he "o&e o the )m is to o"ce a &i()id$ca&&ed mo*i&e hase% th"o)!h the &i()idch"omato!"ah at a secic o/ "ate,e>"essed in m&min◦ No"ma& o/ "ates in HPLC a"e in the 53; m&min

"an!e

◦ -+ica& )ms can "each "ess)"es in the "an!e)nti& == si

D)"in! the c"omato!"ahic e>e"iment, a)m can de&i4e" a constant mo*i&e hasecomosition $isoc"atic% o" an inc"easin!mo*i&e hase comosition $!"adient%.

*asic "inci&es o HPLC 3 5 5

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Sample Injector  In:e#si s+"in!e

S+"in!e diin:e#si#an me&a&)i set)m $sea&#a"et%, in:e#si di&a#)#an den!an #onstan dan*e*as !e&em*)n! )da"a

In:e#si Esto3o/

Saat in:e#si e&a")t dihenti#an sementa"a.Same& dis)nti##an &an!s)n! ada ):)n!#o&om.

K"an same&

Dise*)t :)!a &oo dan a&in! *an+a#di!)na#an.

3 Se:)m&ah 4o&)me sam&e $da&am so&4ent%dis)nti##an #e da&am &oo da&am osisi

&oad, same& masih *e"ada di da&am &oo

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Column.... /ithin the co&)mn is /he"e sea"ationocc)"s

Conside"ed the Ehea"t o thech"omato!"ah the co&)mnsstationa"+ hase sea"ates thesam&e comonents o the inte"est

)sin! 4a"io)s h+sica& and chemica&a"amete"s.

◦ -he sma&& a"tic&es inside the co&)mna"e /hat ca)se the hi!h *ac#"ess)"e atno"ma& o/ "ates.

◦ -he )m m)st )sh ha"d to mo4e themo*i&e hase th"o)!h the co&)mn andthis "esistance ca)ses a hi!h "ess)"e/ithin the ch"omato!"ah.

*asic "inci&es o HPLC 3 5 5

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HPLC columns.... "oe" choice o co&)mn is c"itica& o" s)ccess inHPLC

Types of columns in HPLC @◦ Ana&+tica& @ ID 5.=36. mm < &en!th 583;8=

mm

◦ P"ea"ati4e @ ID J 6. mm< &en!th 8=3;8=mm

◦ Cai&&a"+ @ ID =.535.= mm< 4a"io)s &en!ths◦ Nano @ ID =.5 mm, o" somethin! stated

as 5== m

Materials of construction for the

tubing◦ Stain&ess stee& $most o)&e"%

◦ G&ass $most&+ o" *iomo&ec)&es%

◦ P22K o&+mes $*iocomati*&e andchemica&&+ ine"t to most so&4ents%

*asic "inci&es o HPLC 3 5 5

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Precolumn

Precolumn (beforeinjector) and uardcolumn (after injector) !Protects the analytical

column"◦ Remo4e im)"ities "om

so&4ent

◦ Minimi'e the inte"e"ences

◦ P"o&on!s the &ie o theana&+tica& co&)mn

◦ Sat)"ates mo*i&e hase/ith &i()id o stationa"+

hase *eo"e the an&+tica& *asic "inci&es o HPLC 3 5 ;=

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HPLC co&)mns ac#in! mate"ia&s

 -oda+, most ac#in! a&& into o)" c&asses @ si&ica o"a&)mina, *o)nd hases on eithe" a&)mina o" si&ica, !e&s,cont"o&&ed3o"e !&ass o" si&ica

Co&)mns a"e ac#ed /ith sma&& diamete" o"o)s a"tic&es. -he most o)&a" si'e a"e 8 m, .8 m and 5. m

Co&)mns a"e ac#ed )sin! hi!h "ess)"e to ens)"e thatthe+ a"e sta*&e d)"in! )se.

 -hese o"o)s a"tic&es in the co&)mn )s)a&&+ ha4e achemica&&+ *onded hase on thei" s)"ace /hich inte"acts/ith the sam&e comonents to sea"ate them "om oneanothe" $o" e>am&e, C5 is a o)&a" *onded hase%

 -he "ocess o "etention o the sam&e comonents $otenca&&ed ana&+tes% is dete"mined *+ the choice co&)mnac#in! and the se&ection o the mo*i&e hase to hasethe an&+tes th"o)!h the ac#ed co&)mn

*asic "inci&es o HPLC 3 5s)/edo hadi/i+oto ;5

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Columns " bondedphases

So&id S)o"t 3 Bac#*oneo" *onded hases.

◦ Us)a&&+ 5= m, 8 m o" m si&ica o" o&+me"ic

a"tic&es.Bonded Phases 3

F)nctiona& !"o)s "m&+&in#ed $chemica&&+ *o)nd%

to the so&id s)o"t.◦ 2>t"eme&+ sta*&e

◦ Re"od)ci*&e

#onded Phases

C3; 2th+& Si&+& 3Si3CH;3CH

C3 Oct+& Si&+& 3Si3$CH;%93CH

C35 Octadec+&Si&+&

3Si3$CH;%593CH

CN C+ano"o+& Si&+&

3Si3$CH;%3CN

*asic "inci&es o HPLC 3 5s)/edo hadi/i+oto ;;

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Dete#to" $s+a"at%

C)#) sensiti Sta*i&itas dan "e"od)ci*i&it+

tin!!i

Reson &inea" te"hada so&)ta#t) "eson ende# sehin!!a

tida# *e"!ant)n! o/ "ate

M)dah di!)na#an -ida# me")sa# same&

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 ?enis Q :enis Dete#to"HPLCDete#to" F&)o"escenceDete#to" U7

Re"acti4e Inde> Detecto"

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F&)o"escene detection

Coma"ed to U737ISdetecto"s )o"escencedetecto"s o1e" a hi!he"sensiti4it+ and se&ecti4it+

that a&&o/s to ()anti+ andidenti+ como)nds andim)"ities in com&e>mat"ices to e>t"eme&+ &o/concent"ation &e4e&s $t"ace

&e4e& ana&+sis%F&)o"escence detecto"s

sense on&+ thoses)*stances that )o"esce

e$citation

Mobile phase

emission

*asic "inci&es o HPLC 3 5s)/edo hadi/i+oto ;8

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U&t"a4io&et $U7% a*so"tion

An )&t"a4io&et &i!ht *eam is di"ected th"o)!h a o/ ce&& and a senso"

meas)"es the &i!ht assin! th"o)!h the ce&& I a como)nd e&)tes "om the co&)mn that a*so"*s this &i!ht ene"!+, it /i&&

chan!e the amo)nt o &i!ht ene"!+ a&&in! on the senso"

 -he "es)&tin! chan!e in this e&ect"ica& si!na&s is am&ied and di"ected to"eco"de" o" data s+stem

A U7 sect")m is sometimes a&so o*tained /ich ma+ aid in the identication

o a como)nd o" se"ies o como)nds

*asic "inci&es o HPLC 3 5s)/edo hadi/i+oto ;

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%& ' &isible detector

Ad4anta!es

Hin!sensiti4it+ sma&& sam&e 4o&)me"e()i"ed

Can *e )sed /ith!"adient e&)tion

Is "e&ati4e&+ chea andnot sensiti4e to

teme"at)"eSensiti4e to &a"!e

n)m*e" o o"!aniccomo)nds

Disad4anta!es

Does not /o"# /ithcomo)nds that do not

a*so"* &i!ht at this/a4e&en!th "e!ion

Cannot *e )sed /ith theso&4ents ha4in! &a"!ea*so"tion in the U7 "e!ion

Cannot *e )sed o" thesam&e comonents /hichcannot *e a*so"*ed in theU7 "e!ion

*asic "inci&es o HPLC 3 5s)/edo hadi/i+oto ;9

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*asic "inci&es o HPLC 3 5 ;

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Re"acti4e inde> $RI%detection

 -he a*i&it+ o a como)nd o"so&4ent to deect &i!ht "o4idesa /a+ to detect it

 -he RI is a meas)"e omo&ec)&es a*i&it+ to deect

&i!ht in a o/in! mo*i&e hasein a o/ ce&& "e&ati4e to a staticmo*i&e hase contained in a"ee"ence o/ ce&&

 -he amo)nt o deection is"oo"tiona& to concent"ation

 -he RI detecto" is conside"ed to*e a )ni4e"sa& detecto" *)t it isnot 4e"+ sensiti4e

*asic "inci&es o HPLC 3 5 ;

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Latihan

 ?e&as#an men!aa asa !e"a# cai" ha")sdisa"in! den!an sa"in!an san!at ha&)sse*e&)m di&a&i"#an

Men!aa HPLC men!!)na#an oma*e"te#anan tin!!i, sedan!#an GC tida#

 ?e&as#an )n!si !)a"d co&)mn

PR @ 3 Se*)t#an contoh; a&i#asien!!)naan detecto" HPLC $IR, FRLD,U7U7 7isPDA%