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HPLC System
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?G n r : uffh rIru u r nr :'l#q r u trr?o { HPLC
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HPLfi Gnlurnn I nutu
$etqctan Wasts
filu-l
nr:ldrmnfrn High Performance Liquid Chromatography (HPLC) tdosr:roaouEsunlnfiu?il41 a ja r j er 9 ja y I o dr ; - u 6r
luu[?1flunltuu]Jnu0ut.lttTt;fin1u [u0{0]n tHzuRRl:r9rn'0uililfl-l1ilQna0{ttltuu'lluulluoutunu tu
:vd'udrnn Touu?ri'u{rua^nrniorfioovfi{n^onr:1d.rrurn?0.rfio1#frilfrfmrrurfir1lfinuttnArutfiu rrri{fi0, J a d @ o qeryt^v^
rilrirriurfiuurflunrurd'rnqu rirh,ir{rJflrifrrrufififliyurrTunr:1#{runrsrd{nqu'lildrtJr:rrt{r1o'ld'nsuv
fr,fi'ordrd,rrfiorrur6fldosriraruroqi6nr:rrri'lcJfl[Urrnrrld{rurniolfio (troublcshooting) tLtttilatflu
nrHr'hu ioufiinqrJ:yarrirfiol#{rJ6'ffi.rrufirJ:vauflqrtnt:ldrrutnioq upt-c dr}Jt:oldcjrurfJu
r ru ??r rn'lu nr: r r#irilcuu rrfi osdu lei'
n lvy- | anr:'i'qrirronar:iunirfr'[diun r ru:rilironluou.r.lou.: rtnr{rnrirfrzuouio.rrJg^rifrnr: mr.rrnfi
d^v - r! t- a!1J0{flUU?0UllCIyst:?0flouflfun111flfi?ultlovf.lnfln€uqrflfi?u'r flrJ1'r:drFl: firi':ufiurrilorrov6'qfiildiGnr:
yvr q ntt A o d ' 14 tel d t)u dt , A ' - : 4rtrR t.Uil6u14tnt: [.r{tutnt0.10u6i150on?{ tlJ9l']uo fi0rufi09r11t?t?.1rlJuour{u.1?1tofld't:8lJUu0vt1Ju
','oI S 4 ttr4qv 4
1J:y tuTultnt{n tSilurfl:0.1 HPLC 9l1udufl?:s
Yvfl[uuru09llt 1
d^u - e! e- t!fl u u? 0 u [ | n v n : ? 0 d0 u q at J]'l ?\ d 9t'l u 1 r [a v t{CI n R aJ cn dn'] u I d ry m : d 1 fi :
^d Y\t o4 ct I il?5nt:[lfl tuuturtn-]: tryi tu[9r:o{ HPI_C
9./ a[0ndt:01.:0.1
dI5lJ€U
6ttuilnt:-l{
1.1U1
I
57
9)
l4ut
I
t2
19
22
26
33
JO
41
42
41
48
<AJA
jnt: tn?1
I
2
3
4
5
5
7
8
IO
ilt2
n,d s 4 i A u I q
baseline [rJ [; uu tgruudtlil qrnu?[u o.lfl lJldulll{fl l: [H nlo{d'lT nvCIlu
baseline hiG uu Tor ufi arru qtfi urrdo s o I fl detector
rjratl'vretcntion time tlj0U1.Jl[lJn lfl:O tilqnnO{
grJ:r.rtos peat fierrJnfi
Y4ytat1'9)r{ n n l.i q r u q fu fl r 1 t }1 5 0 11 1.: 9l r u 1J : u r flJ t il q n a 0 n
!4 Gt 9)
Uill'l lfl l: tT.l1ll pump
04 qylltUHlnl: tTilU manual injector,1 q 9)
l-JtUHln 1: try{ lU autoinjectora4 ct 9)
IJIUH lnl: tTill-J column
4 q9/llqillnl: ITi1lJ detector
o4 cl9UCU?llfll: [Si1lJ computer
ifirtiilzuflryNrnt:ldrruornurildo HPLC and CE Principle and Practice
44 9,{Jl ct q y A?fint5ttfl lulJililtnt5 lT{llutn50{ HPLCu
ntlnfi I ?f;uiTltrJmrtnt:'l#rruornrirYrfro HPLC and CE Principle and Practice
rlcynr cIllfigl nrsrrffhlnrAd
1. iil1\U peak 1'llo peak lJ
uulq[nnil]n
I
i'
il ,,
Normal
I
it;l
Itl
---i !-J'.-
Problem
r.r hj16'rilq lamp uo{ detector tiler lamp
sArl1.2 C{lUdruiu-lillryOil91O: v 14 ?'l{ detector
[[du intcgrator r$o recorder l'f o?ilH$o'fl?91
A'El : ? 0 do 1J fl 1: rso lJ 9t 0 I o.ldt udtufuttu
1.3 mobile phuse 'hi'tro qrio z
r.+ hifidrodr wriori'rodr rrdor Rntrnr El:?odouil;ilr[udr:avnrulu viat'irfidalnl
[Y'tu{li{0l.t50 []J
aid6la qdt: iltgr: g ]u?ltgr: ulJ tl1 lJ 1'l lnilu
I q h 'tll 'i ipeak rrdo'nlljfu?i] tu tqOql]lflt 0'1
"!-1Problem
r-J 1? | eHPLC t|grr1Ju]19r?OU',r{U U
Ut4A1.5 nl:n{n111 detector fi5O recorder d{$
[nu [!
.49l: ?0fl OUnl: fl {nl1O.1 attenuatlon ?i :O
gain
2. mobile ptrase 'hi'lun z.r 'hildriJor pump tilotpumptnoO9/vd
2.2 fl11 t14 RCInmr till.t Uqryv{nil : o0nqq{
19r1?'1.1
9t: ?0doilil;illflllon mobile phase'lurdda!|t
cJ ?9t ?t il t1,,1 u.l ?l o 11 : o [1]
nq'-lqFl: ?0doufl 1: t14 0vr.: :vuu?'l9ru mQ Ft [91
' aA q 9) n Igl:'l0C{OU sample loop ?'l}Jd'l lA!11 l1J
q n 6'u u $o fi or n rfl o gn r u1 u r6o hj
9l:?0d0U inlet filter lJO'1 mobile nhase
I n rd?-rdvor9r trJUfll:qqnu
Fr : ? 0douo.lrirj:vnou'uo.1 mobile phase
'i r or ru r : cr n v n r uuh rYu'l d'6 hi rn^sr t{J u
flvn0u
finr: d.gu, mobile ptrase rfluodr.:6
z.J<,i,ailn15:?SN sl : ?0doun]: qiolos fitting vr-r:vul'ir
AUIIua?}Jfi:o tlJ
; 'a ,i, aFl:10flOUl1 pump ?1}Jfl1:5? tnqnl:
fl nn v fl o u I o i lnfi o rf on^rfi us fr o iln n^
A !l I I o 4 dY r.i14 : 0 ttJ tlnU0101tUUfl 910.1[Ufi UU seal
1lO{ pump
-v57-
ctlJCIJfiI cTrilel n1:rrfflu
d I q u i q oi2.4 ill{O{0-lnlfl [U11? DumD Sl tUflStllU0y
rurnrAvlu'ltn?tlJ9ru 0vtlfl ? i t]JFl.lyl
' i' Y ufiOFlll0 1491O[lJ'Ifl U guard column 00fl
xtodr,lrifi suard colun.,n fi l#noqviofrtrutJ
sro[lj]noauuoon 0tnuu nedount:
lu n]ou purge purnp d'ludsr: tnt: r'lun
il:vruru 10 ml/min til,trrfluororlo.r
prime ivlJU ]quld'rruniltfiav pump
[[nviltTvtJUfi chcck,ralve'li?nnru valve
irn',rfr o'[nvlo.ro]fl rfloofl riiileyr rfrr oqj 1#
flush :guud?u 100 o% methanol
919r9los-1{
3. n?riJd'u'lilr'u r?o nrru
#uSrn-irrJnfi
,r"1;,1i l il( './.-.#
Normal i l
/r-l\\\,vf i )\' /\-r-l
Pfdlpn I I
dv43.1 ljflt::?ryil qrio z.:
3.2 nt:'ilinlc.1 mobile phase qnvirbi'uAu
1.1 u9 Tv { n x : on nilq l'11.1qo
qila z.z
g.r fi ilo{ornrelufrr put.t.'p 6s1un: tfi fi ov
turhriY'lU?'lfl?ltJFlu 0Y [lfl ?{ []Jn.:Yl
q{o z.+
<.i,ai9e)3.4 tunl: : ?ryil11 fi tting 9l'lU11l.:[11iJ0 i
flonilu
rloer fitting rrdrsiolrri orn#u fr{dfl:r*i
n15 t?1n10{ mobile phase 1,1 7-2ml/ll;rinrudu4Al4 l
9l?'18{il n15 :?ry}J?l : O l3J
:.s fi r,'lo.rornrfllu : vuuud
flO9l guard column llnUflOnlJilOOfl,
0tnfiu purge iuuu raf ort#rl#la guard
uduYntqtg)column [tOvfl0nUUnnU[Ul t1J tilil fllilruurri.rodl# ptusn TUUUd?uloo %
-Orrunot
3.6 pump seal "irqorlirl#rfrfl nr: frdrL:ou1
14?1JU
njfiuu seat luil
l44. Ft ?'llJ a u 3f 't fl ?']il fl ag
/r.--\\\r\iProblem\>1/
+.r fiflcyrrfi pump r?o injector uiovioqio ft09t guard column ltnvnOd'tJlioonvv
ln
01flUU 9l.1O9l:lfl'l: t?l Al0{ mobile phase
fi z-s *v*in tilnlld'unqoreirafl rraorrdrialvd<
?'l[1]U?1 suard column il t OflOOXJU ilnl:
d'u 1,.{,,,ilnnril{o +.2 r rsitirnrrrrLd'uhiq9)
nqn{'[frrunu]dr14fl fi avaru loruldr?ru
01fl detector lritiouno''uhlfi pu-p
- 2/57 -
rIryur cII119l nr:ufflruAudv
4.2 guard column 14:OflOnilUqqnU9 d xYfiltJ guard column t14[OlOOn 0lnUU
nqn0{ Ro* lmri dtnrrrd'uana{ rrda.lr clr A - qil rA
?l[lJu11 guard column Q9191l-J tl'JlilnUu
guard column ln ri tirn rr:16'u eir niq{ oqj
I di .j - 4 u cl YY s[[dgl{1rljunn0nilu qanu t?lat{ tau
u 9) - t A Ur dfll:nnUqlilflOnlJU [?.|0 tA particle lCIfltA ,yt u J I u a?lOg9ltxruulo{fl 0auuoon 0tu{rn9ta4rrvdu ful.l'tout lfl9t.1?1F]0niluo'10 0v 09l9tu 0tnss9
vl| 4t d u r.t v Atraa
fl 15 UU[U 0U11Qfl ryU0q [UfrOOtJU [9r[lJ1rv6l da 9J clr) j
t'lRlUlU tUn:ilU 0yFl0{ [S so]r'ent 7l
q o a I d Jv.,rl.t tJ tv du lunt: nt09rfl't: uuluouu it 1
u Jv u r Y ri an0A!UUi9lU [14tljRUU rnlet tnt 14]O
ri-Jqlilauunoa]Ju lfitJ
5. retention time 'hinlfi
tiirj'-jU'-jtJL-
Problem
ilii
,i,JUNormal
4ry45.1 iln]::?ryru q{o z.:
s.z dn:ta'lu'uot mobile ptrasc rtldou'hj.,i(nr:nJfi ou dfl T I dxuu oi mobile phase tfi uld I o .r9/
[nnU OUdllJ 1: f]11 1 [1{ retention time
nJfiuurnJn':kirrn)
lu9lT?0d0Ufl l: lJ:ilOn:ldlilUO'1 mobile
o a.i iphase [1Jfl5t]J?l[fl:0{ HPLC Fld}J rr-robile
- a eu v 5 oaq Y 1zj 4ohase [1'1091 [UuFl [14[lJRUUt1JUzud:Jto i
S.: fivlolornrg'lu purnp (retention time 0v
Al1'{lJt [OV OFlttUU random)
lrir,'los ornrfloon 0"rnf? pump ttio
check valve
nJduu seal 1JO{ pu-p lrilmobile pnus. doliltufl1i dcgas
a u /t4 r -i-s5.4 otuilnil10ifl onilu tilniil( t9lulil1nvqa
'AiarOUl.lU.lllJO: yUUlilu ilUU ion exchanse)
cty ii o,ngt 4 ctgrlS column oven YllSOflO [9] 1'l:O lry
ida-tAcolumn mUqU?U (q€UHQulO{fl OnilUm
YleAlaaulfl {Uu 0v tl''l u1J5 y fl ?15tllv'lI 0.1Fl0 O }JU
9/^r rn v 0
-r d'rlJ 1 : 0 n ?uqlJ qiu H fl }J I on
mobile phase riou trlrn od'ru rildff ov 0l 6 )
,ja Y v lao5.5 u?a10{ d]: ma qlt"}non}Ju}J 0'ru ?u}.,-|fl
rfr u lil (retention time ovtiounldrtrnto.tia 9 v ddo a r
d15 vrq olurFt on lJuu 0'tu'lu lJtnrnu n?'r
ud,n?rlJqlonnooilu)
frqrrj?rursufi#ounl niu fie t0 ul rr?rud
ovrflu roo ul rflud'uAurdr
[000'1.191?OUl.1[lJU l: l0 l[0U 1:100
- 3/57 -
rIryrn cnrfiel nr:ufr^ln
s.o d'rvirnynrucrosei?oeirs hiarrur: 0 zudrr
t{rri'u riu mobile phase
rJ fun Jfi €ruei'?ri r ny n ruh.r ri tirril u 1il lpi6f9/ u | 9)til OVAlUgl?0U1.:91?U mobile ohase
S.Z nrod'lrffiilryrr (retention time ovnioul
o ro.:,du o udr rifi orqnr : 1#{ ru u ru fi u )
qe/ - dq 'idt a d - AlTfl 0n]J u t14 tJ?][! uryuqlq ulnu [1^t 0
a /r q v dilq0u'11ilqH'ttuI 0Inn0nilu
6. peak hirrun
liltll lfli ,tril il-'-rt*l uU'Ut,L-----'\-
Norrnal
J'r lilll illr
^'HIUVUL-'rProblem
6.1 mobile phase tdoruanrn finr:rlutiiou
(rflurua1# retention time rilduu)
49l: ? 0 dOUfl 15 [9]:Uu mobile ohase
A v du6.2 suard column H:OnOAilU9lU qffo +.2
peak tnnnt: split z.t finr:rlurlioufi guard column ttio I q{u +.2
ud inoCIlJu I
z.z pi'rriravarutoid'lodxkiarrt:n2oru | rJfunJ6ou6'rlirovnrulnil tirrfluhJ'ld'9 v lqY u I e)l'Ulflunu mobilc phase | lfinvaf un?OU'l{Fl?U mobile phase
lr{t.ILJIUU--lr
Problem
E. peak U tarl Fl.1[[Flt:UIt:fl
vrnqo0na9l
doul8. I }Jd',r55Un?U lUn?oU'l{
d14mq0o{qadl:tl'l9l:$1U 9l?1 peak }J tail
A14r9.rl4rd11:0 tu fit ltju rld9li?tlnq0tndt:
6lura:ufl?u [u9r?0u1i05{
8.2 ld column frpril:vrnvro I - a ^ A ' 4<
AO{ tSflOAtJUSU9l0U ISU n:ilm analyte
df r .i ar c1 g.)cty u drljudl:lJ5vn ouvrlljutud til tryfl 0 nilu
odtYS1lfl deactivated column IilUAU
8.3 ld pH to{ mobile phase uhitrltvaru
rJfu pu tos mobile phase'lrrj tiruildr:il:ynoufirflurua nr:ld pH firirn.r
ovvirl# peak fi nlrudlJiltn TlJrndu
8.4 analyte r4rrJg^fi?orn'u active sites eJO.l
flonlJu
[9llJ ion-pair reagent
tfi ru ar: modifi er yu^nfi tfl uruadarrLr:o
:yt1.1u'i9{
-4/Jt -
rJanrr dl|ilq nr:ufflta4
9. peak tfludulJ'lgl: uoquuridrd
[1J0UU[1-Jl-llJ tatl
d6lvl9. I ildt::ufl 1u tugl?oul.l qdo s.t
AdIAI9.2 mobilc phase [dOlJdnlil Ufll:1JU[lJ0u 9l : ? 0dOUfl l5 t9l: UIJ mobile Phase
aal<ldl9.3 guard column 14 tOnO RilUlJnl:ilU[1JOU
11:OS',l19
q&o q.z
s 14 ld
10. peak [[Un0lnnU [il91
(fronting peaks)
iriijjiririllii,,liltlll.1',-'- 'J'-Norrnal
irlLtl I
;,--U tJ LProblem
d6lulI 0. 1 udt::un?u [u9l?ou'l.t q{o s.r
10.2 peak ffdnutuvrflu'hri uto baselinev
tioun#r peat fidnutuvrioul qw-u orloga d I a i tq-.!,---l^-
[flfl nlno{fl il:v nououY]0fl tun?ou'l{
rfi urJ : v fi vrfr n ruuio rr]fi uu
ft?tlldrlJl5 a'lu nt:#nrfrofl d-1510 {
:vllrdorJ:-1il:" lnt: rrunzuo I peak liio
or o r{o { ldn o dil rfryu^fl du nirr nJd uuv d a ut ,oJ
--^ d1 v 4
olnaonilusuq lilil1? c18 [1JUflOn]JU
yfrqfi{: cyano phase rilurludd I u lao
1 0.3 }J?C'UO.id]5 ilqq t11fl 0niluxJ0-lu ?u
^I,ltxJlnlflu [u
q{o s.s
t o.+ d'r l4 r n v a r ut o{ pT? o d r s'l il ar il r : r} t'l d:J
r{rriuril mobile phase
rJ :-unJ fi uu d'rlir n v n r u'lr ri #rrilu hl 16'
b{ov nrufirodxdtu mobile phase
riount:nnaoil 1# Flush FtoolJuryuq:J
'fird'ru ethylacetate Yu^q HPLC [n:91
dtu'lu 2oo ml 'luo'qr:rnr:'lr1a 2.5t9
ml/min qrildhu solvcnt su^ofi fi rirrjru
fl01.1
t t. ilaru peat fid'nueuvruu
'hirrvoru (rounded peaks)
t t. t rirq.:n'jl linear dynamic range tlo'1
detector
o fl il?ilriu ei'ro dx u $o n r11r 11 uru n {
eranl
t t.Z d'lrir gain UO{ detector srrtnu'hi rJiuir guin'Iilil
d4 g @ dAo
1 1.3 lJ?n10{dl:ilqq[11fl 0niluil0]u?uat4 l
ilrflrnu [lJ
qrio s.s
- 5157 -
rJn;vr drilql nr:uffhA laaA I u 4v
1 1 .4 lnnilnn:ul5v1{?l.lflonilufl ud I o u | .Y \ .t
o inilsvnou [u Fl']ou'ln (01?]:]u tfl :{rT:1.:
d I d tcl u I I
10 { 0 ifl il : vft 0u11 0q tu9l?0u-l.l 0vry1unl9l
a taaa I4 t.r9ltirl: lfl 9rilan: ul [9] )
rilfi uu grrtrL[t:.tllo{ buffer, pH uio
dfl :tc{ruto{ mobile phase
alriluqiuHQlJl0{n0ailu
t4 a a dlilnuuryuq10{noa}Ju
t t.S d'{fir time constant 1Jo{ detector u$oal4 l
recorder d{[nu IUa
si'seir ti-. constant pirafl iliorirfi ovfi rcr /ur Y ia -i[il t9lp.lR14qnq9r
12. baseline drift (baseline
u ,AilJu{ )
Normal
Problem
r 2. l q iu ?1 r"r, 1r^ u 0 .1 fl odil ft ri nr I fi t r r t1q nr r 4 ru^qos
rj d d Y do49. .. 4'A q
[ilauulilu{[anuounvll [1'l baseline [lJu{ tu- idr ' JA alnn!ils?ltilu cycle) UoUn: illqfu?lQlJfl {t{n
tqduon:v 11u9r09rsun1: fi fl[14lJO.1[[dnttAenlS U1
lr'l$lrto.t detector triu uv detector fifinrrrut4 a ctY 6l"[rar liiont] ['f {1u lu:vilu indirect
photometric rnode
fl ?u qlJ qfu ?t q l^r-r o I n odtL dt tov rnobile
cry 1 d riphase [[0& 010 [Sqiln:il[[antUaUU
o il il n lJ^lr o i c{ I : n v CI I ufi ou rrlr dcrectorj o qY ;
[v{0a9 qiufi Qil1 0{fl l: cvnlun{ t?l Fl{vl
i 2.2 rr,obile pnase'hi rual rrsu rfr o td uriYu
o oi e
tlun:tfi fi baseline rinovfi d'nuorsi.rffu lri
tflu cycle)
'ld solvent sfre uPLc tn;91
dr:rnfifildt{iu buffer rtflv aclditive c{ol
AA*iln?1xJU5q1]5q{
degas mobile phase riouldsru
1#fi'rry6rdurutffo degas mobile phase 'lu
I 09:Y1'1?1.: [5.1'lU
I 2.3 detector cell dnil:nufofi l'lolornrg Flush detector cell d'lu methanol tlioa{ ia I
solvent OU 11}J 9l?1tJ It: { fl { n X]
l4rntrtavotfl detector cett rTlu t N
Hxo, (r?rrLl#Hct)
12.4 outlet line 1Jo{ detector qoroi'u dr1#
n?lild'ulu detector cell {'1 rflurrqt#
detector cell window tlnn#l? a.rrua1rl
baseline hi6uu
AJto1d.11'lqqglU outlet line 1J0{ detector
oon
trjduu outlet line 'Iuri
nJduu detector cell window'luri
12.5 flt:zudlJ mobile phas" 'hi6 riodsr:rrn rjn]: t1.1RIUOUU
Yala : 1 0 dou Fl?1}J 0 fl 9t o.l lJ 0 { 0 91 5 1 fr ?U{
AU14mobile phase 14ioogl:lfllt tiln
- 6ts7 -
rlcymr d]rfi9l nr:u#l,u
tz.o nod'ru#uhq cquilibrution dt Jeutqilrv
odxOrrfi ofi nr: nJduu mobile phase ru^el
hiri
u dY iaFlush fl00uU9l'lU solvent ?luil?lu[[: I
!
rJrunax ttnuiu flow mobile phase Tu^Fr
ln rirJ:vrror 1 0-20 rvirqo.rr]?rurpr:udl
fl0nlJu noufl-l:?19lflou
12.7 mobile ptrase 'hiavorpr rforfiolJdRt?\adoia;
1.1 : O [9r r UtJ 0 1n fl 1: lfi]J 11 ]J fl [Un lil 9l'l
d
9l: ?0dOUnlS [Fl5Uu mobile ohase
a j 4 a 11 v J4tI 2.8 udlT 11d1il t:0u9l9r91 0u tun0nilu[1Ju
t9
r?n ru ru 1t<' fi rirqr t oqjlud'rodr r 6.rar:fi
0&ofl se oofl ornnod'l$iludns ilvu 0.1 peak
A gt I o q yu j 1ytfl ?1{ou't{u]n m] tHanuilvllu:]nfl
dctiAAoofl U-l03l14utilu baseline 11?n{n1U (fl 1i
vv
run t !1J u gradient ri rb?rn^a fl ryx rfi rur nn^u )
'l# guard column
otnove{o{ Flush nrodrurirhu sotuent dfi
n?rurr:n'lu :vH'itsnr: fi eeirodrr riorfl u:vov1 lu:vv'jrtnrs nerdou
tz.s finr:dr mobilc phase no'lttrldlu:.i trsi
hildfinr:rJiu detector
baseline lrri9t.1fll
#rrirq.rn'jr dynamic range 1J0.1 cietector
1#1# mobile phase fi rn6uil1il :.j
rn rhe./u i A A {I 2. I 0 []J lfl ?91 d]: ]1n?tilUt?nnUil d't:uu
drilr: CIqlqnfrurra.r 1d'q.: qq (fu max) ('luq
n:ifi fi 1# UV detector) rrsiiqar: fi n'lruu-t?I
4 4 u A \ttYg)nnum dt:uufl rtj'rr0qlannu[[fl .1 [9tu 0u
ridr4dulil nuu[1Ju R ?1]JUl1fl RUl',r dl: UU fl t]J 1T 0
a \19/qqnaurrfl 3'l0rq r qn (), max)
13. baseline hiGuu lqufi-A1oonHilvilun9l
Normal
Problem
t :. t fi r,'l o.r otfl '1fl 'lu mobile phase, detector
cell r$o pump
degas mobile phase
flush :suurffolrir loroinrfloofl orn
detector cell r$o pump
a,l,*13.2 runt::?ryru qile z.t
I 3.3 mobile phase f.tdruriuhiaruq:af 1un:fifirn$o{ HPLC pldu mobile phase
ct 9/u s -oq Y liriloa rulJgl r14lunuutilu p{dilro I
a 9 dd A 9)
lY solvent Yliln?'lu1'l U 9lU0 Un i
I 3.4 zuan5v ytu0rnqfu?t Qfi (niu d"r qruxqt-
nod'ru rfliq{ r rsi detecto, fi qar r 4n^ri r;
talnqfl?tlJttnnfl1.11J o{otu11n}J: v 14 ?1.:qq
column flU detector
qy I d td0tt tToufl : [u[lCInt! nuuo guHnilu0 iq9{
,\d]: nYAlUflOU ['lJl detector [WOO9l
o qy jo€u?t n il1 0{cf r: nva'lun{ til n{Yl
- //Jt-
rJcunr cnrlr9l nr:ufflr
r :.s 1#lv,htr aru16 urrYu qrJ n: cri'ldfi rdu 1 [[un LC. clctector. computer rfiully'htrri'aq
fl uny dlu 11 r09t: ? 0q?1rfl 9l 1Jrufi t0tn14 t91 4 l4 'tl''lv{ lnluu 0fl l.i : o [tJ
I 3.6 d'sr rvnl: iltJ d'r: avnrufr srrjn6AUUA4fl 9lsr { 9r?1.1 U ?{ 0 i14 ?V f,il: LJU ( pump
. ul 9/odampencr) [? [U:Y1]1J
14. baseline hi6uu lFtufi
-.ioranfl!fljvmt,t9rufl9r
Normal
\ewProblem
du414.1 ilnr:51rytu q{o z.:
n, a A14.2 mobile phase llJdY0l9l 14sOldOlJdtl'Il^'|
adoia;1.15 O[n: Uil fl1fld]: [n]JnUq€UnlilEll
d
9l:'10 d01Jn1:tFl : UU mobile nhase
14. 3 tfr q0tn:vurL'lvlfi ru o{ detector 14io
computcr
t tufl detector, computer rfi uu'lv'hft nr u n v
fl'lu
t+.+ fi ilo.rornrfllu :suil Flush :zuudlu solvent lifi n r trurt:.t
t +.s fi r'la.ra1ntfllu detecror Purge detector
AqAUVInAn.: !n5 O':'lqFl11!9lU 9lO 01fl detector
I
14.6 dcrccror cell dnil:n (nr:rjurflourfiuld Y d" qgt- .. ll,a tlt
lRflU0Uillll [1'l baselinc t]JltUU ttl )
viln':rru av o'1fl detector ccll
14.7 lamp 1J0.? detector tdOlJ nJduu tamp'luriaa9,/ A @ A
'q @ J
I 4.8 rynnril:0?dE?] 0u Iun0a]JulJr{fl'ru
finqoofllJt
ri - Jqlilnuxinonilu [fiu
15. peak fid'nuarsnirs
(Broad peak)
I'rili\
#l'.lLJL-Problem
ts. t d'q:rc{rilto.l mobile ohase trjfi uu tn6uru mobile phase luri
ts.z dsr:rnr:'hotol mobile phur" oir
al4 ltnu tll
iliudn:rnr:'lmn
aLa.s'Al'a15.3 ufl]::?ryil ( lflUlQv'l]v0U1iUifl]::']ryil
: v H'jrsfl od'tJ dri'l detector)
q{o z.:
ts.+ r;t""rnir detector hionrior tu u I al I
lJ:1jflt56Nfn tl.ltJ
I 5.5 zunn: Y 111JUO.t extra-column
io I u d46a. lJ?ff 1JO.1d1:mAqltlfl OAilUil0lU?U
avll}J'rfl[flU t1]
qrio s.s
b. response time 1JO.1 detector HiO
il?:L t gr :t o r detector cell a,-rnlfi u'Iil
6t9l responsc timc uf old detector cell d
16nns
- 815'7 -
rftymr dltfiq nr:uffltI d | | v lu
c. viofr qio:v11?'l{n0oilufl u detector
A h l 4 9t t d Gl Y.
ur?rilu "lilil5otdu zutquunnl{n-]u tufl ?1i
lnu [u
ldviofi fi rdu rirqutinnrlil: vtLtsu 0.007-
o.o 1 o d? 1#du fi aetrvirfi ov fi r'ld
d, response time tO{ computer q{allt
rnu til
091 response time
I 5.6 n?rxJtdru'ilutos buffer riourfi u'[rjAye)
ty{iln?1}J[1JU1U
I 5.7"guard column anrJ: nn?odrqer lilnUU guard colulnn
1 5.8 column anrJ: nr?o'f t:"et
I
I a r I a g ,Y- -4I i s.g rnnvon?1.11'lll1'lt1Jlfl 0nlJul.
I ts.to dr:il:vnouulfln?l 1 sun QnsvoonI
lvlddI olnnoniluvl[?a][9lu?fl uI
nJduunod'rLri drnu'ir16' p.ut d rtnutvd't
d?u rtdet{ ?1fl onilu rrilfi fr .rrlurfl ou o?'r
1#dr r o o d'rL rfrrirsr rrLTG n t : d tv ru rv aru
rdo ov rirndu ru r1dld'lr ri
rilfiuuno6'l#
ru] fi u u ryuln zu o { n o drr d. fi dl u h{rfi qr n r :,
rlufluoidrr"tptFltu
I 5. 1 I qiulj Qn^n od'l riPirrnu hJ rnaru qiu m qfr r rri hi n r : q'rn'jrd'x qaru qfr
fi no dru ririu ar rur : nld.r ru'ld'
16. n?llJq{1oi peaku
^4hlorulu I peak lJu tlJ
nJduuurjas
I 6. I dr:ilTvnolfi oqi1u6'rodrs'ilruru
ar: du hldolr dn r?.l u6o n odrL rfrdo ru
fi o ar: av n rurirodr lfi rsr? urLlH riHiofi er
aia-io4Yd]: lj'lFl: g ]ulltouu uu?lu'11o{Uqil'1 CI1
Auvt)peak U1{ peak 11 5 O11.1r ruqUnn{ g'lU 0U
,i,dtqYli-d"tts)niTrutovtilu liltilnuunon]Ju t1i]J ol
v dq 'ocle/ +4- q-Yfl onil}i [H iln.] t1.1 t{nnl:1191dougl1ju tl"l
s) u d I ad j . 4-6rsn onil u tntntil ?r n1: u [?1]Jleflu [t'10
O 9'rl4 Ydl
flvulnauill [s t9l[14il
oi'asralt6.2 fr nr:irryil [qutqnru0u'l iu{:v1'l?'l I
v e - dq- ---ed^^.injection port fl uyll.l[ttnonruu tun:iuu 0v
. ri rn ryT,lUil retcntion timc 0UtilAUU [1J9?U
q{o z.:
4dt4!-4I 6.3 iliu'l9l:91?ou1{mqq tlJfi ill er T ? 0 do il il6rL rsr : fi fi srir ns fi n6o'hi
s yqv -tFrUo'l IT sampte roop triorrJ?:J'l9lTfllfi
h?fi qr ar : o v ai uei'rodr l'lu rj?rursu z-:
rvircrosil?il1el5 sample toop rfi o1##u1o
'jrar: nv a ruri'ro dr ll : : qoqinru'lu toop
ourfiil
- 9ls7 -
a,lJ6ufi-i crlrmel nt:uffh
e| : ? o dou il;ilr Fr 5 d']T n y n i upi'rodrs 1urddahr
vial ?1Ut1\UAY{O14 5O ltJ
er : ? 0 doil'jtfi v'l o I ot nt g1u syringe-type
injector uiohj
el : ?0dou'ir'lu,flu fl ounr:dr.:u$o flush
tuuu solvent fi 1#hidrl#at:il: vnou6lqr4lu 9t ?o u1.1 lfl 9l fl 1 : 9t fl n y nou
Yruld4
16.4 n'l:n.:fl'114 detector 14i0 computer
rd h lrq lAgrq I lq I6U6IUH AII
al9!:?ndoilflt: ainl
-!,
16.5 lamp lJO{ detector [d0l] nJfiuu tamp luri
16.6 detector cell ffflil5n litnruavotfl detector cell
1 7. n11ild'rur:n'lunr:rrunrihr
peak t1JfiUU tU
i
I
I
itr,llllt,rl I llllf,idt',JvlJvL__Nornral
I
llI
ll|-},L_Probiem
d^dUA1 7. 1 Unl:11^{XJ14 : OAnf}flJduUfl lOn solvent
Itty I
t9tfifl
dr ll}- n?]:Jr[: ilJO{n ]5tljU [000U
- nIJv--vv- n?ltLlfl]J'lJu'Uo.l additiu. fi1#
s ' A A qy- q I o ilqBlq il1v o u1 { u.1 tu o tsf}1J9r?o u-t{ su Fl vl
<t lntlju tooolr
9l:? ldOlJil-l: tflSt,il mobile phase
t z.z fi nr: nJfi uunod'rrfdu ln ridsfi
n ? r il d'r tu'r : 61 1 u n r : t tu n peak si t I I t n du tFl^tJ
u 4v a t t
9l: ?0dO1J packing 1J0.1n OnilU0U tll:J?l<1 ^4a4l4r9/tuu su 9l [9ru']nu 0u [9rlJ 1.1 : 0 [u fi 1
olo rn r: l#klzunn r:uq aor tu d'numya Q9., ry / a a I J
t9DJfi?5 tTfl 0ailuryuqrqil t[9tut.:fl :{uda4vrgra
n oa il u ryu q tfl u?fl u r r9r fi u c v rd r.to9t 0 s
GtY - | u L,lr)[1'lt{nn1:tlUfl fl O{ neak ttglfl 911{fl U [91
aa' 14 '17.3 solvcnt 11 ttnvnlU9l?OUl.1 tru[?llJ]Vd]J
4dutiH:oann?oUlnmaYnlUlu solvent ryfiqfi fi
qtaio4tlFr11ilrr:.1XJ1n tUil5ill6lt?lulnlfl U [U ISU
dr 9)
100-200 ul l1JlJCIll
rJ :-r nJ fi CIu ei'rl4 ra v atulv ri tlrrfl u hJ 1d'
l#oy a r upi"to dr.r d'':u mobile phase
audtaVttI 7.4 oilJ?tniluo{nonilutU nuu tljgq rJ iu q nr r q ru^cr o { n o d'il #lil il
r7.5 nod'rr#fiqelrjv?
tuauunonlJu
- 10/57 -
qlilAJfiI cnrfiel nt:rr#h
18. peak nd'urir (negative
peak)
il
.*lf,-it--'-Problem
-,$
t s. t lriarur6oil siocr or n oru n^rrn o fn n''ud'r u A:?nCf0lJ polarity
t s.z 1un:ifiro{ Ri detector ororn^eltnnr:idrylTl refractive index fl O.1 d'l: il n lU 0Un?]
mobile phase
qy !a r
tT mobile phase 14}JFl1 refiactive indcx
io9l1A'101ntqlJ
u9/4r^dn n uq tu uo.1 dlulTo u 9l 0 n o }J?\ ?[9to :
tS.: lun:fito{ UV detector oiorfiqornd1 u o v I
0 i fl u : e n0uu 0 i9l ??11 ny n'luFt ? 0u].1[t Ry
4rurmobile phase lJfl?1lJt [n nfl ]nfl U 0UN]Jln
-lY f4 u o q I Y 4 h thr/u:uluauuFtllltnyatu tuil 0tlilu l1i tgl
cll 9)
tfi AY nlUFll0Ul.:Fl?U mobile ohase
t S.+ lun:ifi fi hirJ:rng peak l1o.ld1i
6'rodxluln:ilr Jnrrfl iil ororfreo'rnI
rnobile phase Qfl nfru [[d{fi fl t'ltJurrnfru riut49/d I a I
lqafl?]Fr-lourn
r.i dr q9) 4i jtU nUU tUU t$fi ?'llJUl'lFl01l ?1 mobilc
vuhtAphase UIJ tilqAnnut[?f{
nJfitru mobile phase rfror1rilfililai
Bn nAu I rdifi n?r! u-l'tn fi u li1#d dn I do
19. tJ peak 1l tlJR?JIJ lu
solvent (ghost peak u?o
carry over peak)
+Normal
{solvenl injecled ailer sample)
f,/I-fr,=+\Pioblem(solvenl injected after samPle)
t s. t fi ni: ilurfl oulu injecror uionod'lri d
Flush injector Iu:;ttlt.ti]rlltftd01J4Aiodr ro
luu diytn'l:ntr!uilt s n1
I-UY
Ai-16rv4lTl0Tv fl 1: 11 ?f
'llJl:itnnry! o g tua0auual 9)
[Ft]-t tuo0it
Y a dY ddnl Sn0nLUFl?U solvent 11:Jft?llJi[: i]Jln
lu n: tfi fi mn gradient lr?rfi triusrounr:gtgtAi-l
n1{ gl0li-lu rl^t 0sv dt:ytd-lilt: 0 gnryuou
lunoii'u#tpi'uruoofl
- 11t57 -
sp:t$d 2 - sp:tsd tz r{lu{oryaornn#rdo Guide to successful operation of your LC system
i u r"r < d j A - .z-.---.---q.gt't:t{fr 2 baseline'lritiuU }oufrOttnqtnu?tilonnill6iull'lin1: lildilo{dllaYAlU
1lfu11t arrilE nr: rrfil,u
L baselinc hiGuu ludnusuvirn ' dr11 [lJ[1J1-J cycle
,lEl6lfl<l
tNormal baseline
lr,l Jl:l I tol , I t2>-4-r
t . I fi l''losorntfl uu'l9l
luqjoqjlu detector flow
cell
purge detector flow cell
rfi lntrrd'u 16n#oufi ryo{Tt'1oofl 2o.1 detector
degas mobile phase r?o sparge dru h"liu-
r1 o r iiu n t : tn^o v,l o I o r n I g'l u fl ow celt ] qr usi o tube 1J l-J 1 el
v
rdur"irqurinar{n'lu'lu 0.009 u^? (0.23 :l^afinrn:) rray,
rdu rirqu tinor{nruuofl 1 I 16 fr1 ( 1.5 8 tl^nfr tiln i ) ur? 1 -
3 {or (30-so twufrrln:) frryosilr{oofl1lo{ detector d.l
tuu" fi o v d r r rir fi rij u st'"1 d r fiq n r :'l il a u o.1 dr : n v a'l u,,
tflurual# backpressur" rfitdu lqu tub" fiur': : rlEr (qo
lryu91^[ilfi:) ovvirl#rn^q backpressure 30-50 psi (2-3 atm)I'o
Air bubble in fiow cell
Tm
Unstabitized system
E
Tm
Leak in detector flow cell
rfi o lddsr : r n'l r'ln un u,,i', I mliu t ti o d r.: 1 : fi sn I ri ou
, J "t - I v , g- r- . 4ivFlO tube ut{tnl detector 0vglo{Q9llU?l tl'lFlo tubc U
rr#r ov lilli r1#n I rrL6'u qln'jrnrrrud'ufi fl ot" ceil ton
dctector ryfin RI, fluorescence, conductivity ttoU
eicctrochemical vlumlu'ldd I rdl
1.2 ilvi0{0ln1flou[u
rcference electrode
([QYl I Yelectrochemical
detcctors rvirriu)
d I refercnce electrode o o fl lJ 1 [fl'l v [u r 1 rrrr^o'l ri
vlorornrg
tlrtirn r fl sur rhi'nj fi uu electrode'lu il
t.: fi v,lororntfltulq[6nzi i, 9)Vtq
lnn0umpilu[1Jl [u [u
rduvn.rnt:'lilouo{
d'l5ava'lu
anrtprime pump [Tlo tny'loiolfilfl
A9degas mobile phasc fii 0 spargc 9l?U heltum
1.4 :vuuti{'hirafru:
il?oti.: hi equilibrate 111{
tfi xJ
A og,lr | ' I
rfr' I n n r "[f
a ? u il : v n o u gl 1 { 6l u o.1 : Y u u [ul-l column I tff 3
dl
detector I n f f ru tdn ui [ [0 u equilibrate Yl'l{ lfl lJo U'i{,9
tfiusilo lquqornanrtvfild tryu mobile phase fll5d'.1U-
ra49)fll detector tlnUTUfl detector [UU9lU
1 u n : tfi fi run gradient o v d'o I l#r'la r : v u l n oiu r{r {ti4utvtt4 I
equilibrate fl OU?'l0vqfl fl ?0U1.1fl ?n0 [U
- t2t57 -
1J6U1.11 d-l[1.rE nr:rrfi'lcJ
1 u n : tfi fi 1 d ion-pairing reagent t v'ldtrn r t tn s rJ?ru r ru
solvcnt druruuff l1ufl1i equilibrate nod'ruti rryu ld
dr:nvnrul{ru{u s mM dru'ru 100 ml fidEr:rnr:'lvrn
I ml/min rilur{u
t.s finr:rjurtloulu
mobile phase
rsri uu mobile pnure'Ir ri ler ulda r : rn fi fi fi qru n rr^r 6
tloy solvent Yu^91 HPLC tn:F]
yirnrrruayorer filrer fioqjluzuroruTtQ mobile phase leruqeq
ulf19)iU'l filter [],J sonicate tu 6 N Nitric acid AIUFr?UU1 3
vu9rfl : i [[nYfl lSJFl?U methanol
Flush [[0V equilibratei Ulllr ri
d,i,ail -6 lJftl:5?SlJll detector
flo*'cell
riJ q t l t n ; o u detector q q or ir fr r tir r o.r hi rfr u'l #rJ n rin^
-:9l{u
1.6.1 Flush detector d?t; sol,uent dovarutrlriYuld']nuh rd dr r
I"[:.i fr uuner rfl u ot u ru al o r n fu sr rrL d'?u Methanol
t.o.z trjrfi'rry nirrogen m?o helium rqirhJvrxvo.rcr9
inlet lOi dctector T-11 0U florv ccll [[14i
t.o.r tlr baseline Guud udni'it rnqnr:frfi detector
flow cell6t9)t a rit1'l cXOlJ[[ry]J?l : 0lilAUU detector fl ow cell
dtdl 41.7 iln]:Uu[lJOU11
noailu
eri ?odou]qullilufia oa''il " firuriru union niooro'ld
tube lrurflrdur.irqurinor{ntulu 0.009 fi? uml-J unionA q Y d @ a qY u q , !
rvto [l.ttyUU]JFl?']tJFlu riold column duluru o'lfluu
run rnobile pnur" 6nn9'rtdoq baseline
- tir hi n u il 6U 14 r 6 n r r afl {'i r fr n r : rJ u rd o u fi n o d'rL ri
rFru o?r b{fi .rriouJ 6uu n o #ruriln ri
- dr il rg r r ri': n ufi o du r rd q n'j ro'r 0 rn^fl o r n n r : fi
solvent'hi a r rL r : n zu ail rcir #u 1 d r io fi n r : rJ u tdo u fi
mobile phase ttio guard column r?o filter'lu:ytttt
- 13t57 -
o4
lJfU 14't flt[1.18 n r: rrii'i'u
2. baselinc kiGut lud'nunrvia, q I J11[1J1-J cycle [UT?i[?nldU6l
(Turvr0 ru tvr)
b
lkEAir bubbles in detectorflow cell
Tim
Inadequate soivent blending
Inadequate solvent blending
2.1 n?lil9lU1JO{ pumpd
4ua414: O0.1141v n',l;lJlJ
^tadt:avo'ruzuqunFl
9l 5 ? 0 dO 1J fil : lirs r u u o I prcssure transducer rfi o'lrj kiL4odrJd.l fl ow fl ?: tJn?'llJFlUtlJU fl U U
Primc purnp'iuri
fl : ? 0 fl o u fl "l : d r s r u cJ o I p,,-p'j t li r I t u'l dvt-.: o o I pump
at'lr11:O trJ
dr'ld rolu"nt d:yrru.{ru 1# a"nu, lnri9) dt dr A r^;tit til u n t : run i tull sradient o v riju as il nsr fr n rIru li'u lri
itra a i ri r,rri u I
fl { il ry { il d 1 [1.1 E 0',] fl fl ? I tJ 1.1 U q m ril n U U [ ]J []J 0 0 9l : -r dt U
,i1Jo.1 mobilc ohase tilfiouttilns
gttl,,,f^illgl^ilfifl1T'1.:fi I 4g s
2.2 fifirtctu solvent '1il6 et : ? I d o u'jr il q r.r r rfi or o r fl n I : zud:J solvent ki 6 oi.l4 14 'Sfi50 llJ tnu
- flow mobile phase A 100 N dlulu 5-10 tvilclO.:
f^udutuTgrSf,roouu
io- flou,mobile phase lltldU[O{ tUOglSld?Unl{",) IYU
s0/50, gs I s, 7 alo u€oruausr rrunr: ldrru r?r9J du d o r.ifi'l baseline 01nn1: run 100 % A ilAflHfUvt5UU9l ilFl[]JO
I v | | 4u 14 rd r
mn 9l?UOfi5 ]fl ?U911{cl il nfl U filY tti[: U1] tlfr 9l { ?lnlt,9
rudlrsolvenr fi il rum r o?.: 6r o r o fi ar tfi sr q-l fi:
2.2. I solvent fr ldhiairu 15crrudrurdrriuId' i#ri tr r.tg/ A y u q9r'4
tUnUU [U [Y solvent flzudtJttlnU [9191
z.z.z finnufi qilnGfi pump tnu rrdtff ldrJil
dn : r a ru solvent u ?o o rJ n : nffi li r r rir fi zu aru de un r r ru
fluq{ry1qet
2.2 3 n1; wdtJ lJ o { d-} : a v o I u}J dn o o n o r n pump'} ri 6
qvA -:tl.t [1.,i]Jnt: zu dtJ1J0{ dt: ny n tu9riu
o 5eH'j'l{ pump outlet Il0Y injector hi'siod'ru
tube lllrrflrdup.irsudnaxnrulil o.o+o fr: urrv,9
az{otdt4rocl/6-12 u'J ry{n1511][TUU0v IXJVn [?rfl'rT run
gradient fi q'l il o r n rrl^il
- 14/57 -
1J ru1.i -r drrlJE nr:rr#'lr
. tirnr:1# tuuc'hiorrur:orulfl cyrr l#rn^ru
I d io "
,i s a ny I
01jfl :ruzudlJilu1uI9l tr4tulju tqunq t?:v11 ?tnr 14 d ng)
pump outlct [[0U injcctor OUI{ l:nnlil n?: tT
orJn: srfe.ralfi fi r u rprrdnfi afl ryirfi tvrfl u hJ 1do, ,
o
A9lua[1]O1J0{nUn]5tlltJ delay volume tt0Vflll run
gradient fifrn;rJh.l
2.2.4 bldU solvent [O.1
2.3 tube firio pump inlct
AHn1}J.tofi:0qqau
er : ? 0 d o u tube dr u a r ru 1 #n ry u 1 dr nj u tir.: o l#d r 1 #
sr : r tYrfi nr: qori'u l#nJfi uu lu ri
z.+ fivloqoinrfluutel
1r€1joqj1u detector flow
cell
qdo r.r
2.5 pump inlet chcck
valvc dnil:nr?o"frqn
?llfl?llJfl YOl9l check valve
tdra9l : ? 0 dO U ?lXJ n'l : fl 5 0.1 solvent 0 Ul {tl'l }J I y dlj [1''l O
ilv-iUO{nUfl l: 09lFl U il check valve
a - j rnvAOlnlilqnl:Flt]flynoU1O.: buffer 11 check valve [9tlUO
^idr ' f, -run d I 5 0 V d 1 U ?1 [lJ U d ? U 9.1 d ]J 1J O { U I n il organic solvenlvvv
lvtAtrttq4dtu 0 9t t I d? u 50y, clu t1J vt i u n I : 9r fl n v n ou 0 y tJ il I n
Ld 6l ,4 Aqr)[1,',1U.1 t9ltuUOufl1j$u9t1o{ solvent ilOU buffer 11 l'll$
2.6 pump plunger seal
A[doildRl1,{
njfrou scal'lmri
2.7 solvent 01fl detector
outlet 14U90{'lutreq,614
:0{:U waste ( tUn:0Jia n
1JO.1 detector TlJn?lll [?
tvlFlofl 1: tila20{fTt: nvnlu
rvu zu rtav
electrochemical detector)
'fi'q il o r u vi o fi si o o r n derector outlet'l #ii'ru riari'u e.r ril,u o.l
- A qgt h9/141J?91:0'1:U waste 11'10 tl4dlSnyAlU [9] tHn0OflOul.l
ri o rfr o r hi 1 #rn^e riJ u d'nu il v n r : r ufl o o n 0 r n il a r uvi o
- 15t57 -
o4
lJ[U141 flt[1.191 nr:rrri'lzu
3. baseline hiGuu luo''nucuyid, d t
?1[1JU cycle [Ury?.:t?nlU tU- aa o! s(utl1fii5? til{)
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3.1.1 1# column heater fiorurnfr s oC rufroqa
^9)q6u?1QlJ11O.:
st A rr 'hy.l y j^3. I .2 6r urnto I
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n{?'l
r^ar):. t .: odr"lfrnr?olri'rufiarrd trreler
:.2 finr:rir solvent otrl
detector wastc outlet
nd'rur1#1uri
l4r'hinr:ril solvent 0'lfl cletector waste outlet n#urLrldcl I cty j[?l u n?5 [s solvent mrqr? uru lu rilroy e^itu nt: n: o{t[d?
,rvirriu
4. Baseline drift
zf
Tire
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4.1 :uuilri{hirafiu:A ul0 |
1'l:0Ui llJ equilibratc ?11{
tnxJ
q{o t.+
a9 n I ej4.2 0fill.inil?i 0{ t}Jfl i?'rqq q{o:.t
draro4.3 ilnr:ilu[]i0u tu
r --=^moDlie pnase ?1) u
td0lJdRllr
vquo 1.s
4.4 f1ll degas ?{ fO sparge
mobile phase hitururvan
dr mobile phuse 'Iil degas ttio sparge drg helium rtdr
ri r'h I equilibrate 5 v u u 1 r ri dt fi eio I : y i.r o dr 1 #n I : li'l
helium sparge d rl#d'n : I a? u I o.1 solvent trJ 6 uu hJdv.{d
4.5 URI:5?S]JY| detector
flow cell
q{o r.o
dratd4.6 iln]:ilu[1j0u11
nooilu
q{o r.z
+.2 fint:ril solvent olfl
detector waste outlet
nd'urr1#1rri
hinr:rir solvent 0tfi detector waste outlet nd,ururldq_--!-, cl9/ i a q | | 9)[14 tJ Fl ?: lT solvent yllgl : UtJ t1.l tJt[ny ru lUnl: n: 0{t[A?
v
tll1UU
a o!< o4.8 Ufl]:5?SUU IU:UUU er : ? o do u fi tting vil fi u et t1tnr I n r : fr dru fi q n 1 or fi 1#
- -qy | , u t6ty I a t4t.9u "i 'HryU t?ruuu (5v?,lout tl4[[uurJtflrnu til) otu{Fl.tr?o(.1
l#nJfruu fitting tray femrle 1tlri
- t6/57 -
1l fu14 I flll1.1s| nr:Irri'lr
4.9 Stationary phase
I
u'r{fl?uilnqoofl01flq
flonlJu
et t ? o do il I n ut [m u fi fl o dil rirq^tJ d'r u union u ?o o r r'l d-Ytdv
tube uutqtfluzu'lfluunnt{n"tulu 0.009 u^? t[14I-J unionU
.i qy 4 q e Y
['l'{o t}'ltYUU}Jn?l}JAu O]n#u run mobile phase 6nnfS
[?',lOFl baseline
a : ? o d o u'j'l a n r : v fi 1 d.r r u ru' n r r ru ru ru r v a ru ri'l rr o d'ru rf fial r 9) u utg.) | jIY[YU nl:t.ldill1lflU t9l1J0{ solvcnt S?{ pH [[AVOilcly iq rt J a dq e)0lfl nl?u lt t5{]uu d{p.t Rgt oFl 0 RxJu t14
o ttJfiuuryu^Flto.t mobile phase
. nJfiuuryfreuo{nod'uf
4. I 0 n ?'rlru'nnfru fi ld1ri
tl.lUlvdlJfiu mobile
phase
ayA9l : ? 0 dO U fl 'l : I I n CI U I t d.? 11 0.1 mobile phase O ? U lfi 5 O {t
Ytddrsspectrometer fl I 1 l U ?'l n I : q n n A U t [d{ lJ fl 1 d.l t q Uq 0 I n
irn' o qy, ,- c3r d tly | 6lnl:l.i llJd'lUlTCllll [?{ baseline [1JUf,UU'l tfl tldAill lU
mobile phase fi ar: il: y noufi drlJr: nQqnfi uIrd.: UVd oq9/a -^9 4 , Jnr e)ut s0ivl1tl.i[nq baseline drift nllilulsuu tl.ttln tt tFru
. nJfiuunrrrLurrnfiufil#1#finira.:n'ir uV-cutg
v
off 1o.1 rnobile phase tiu';o tilnuuyuFluoi mobile phase
4.11 fi stabilizer OQlu
mobile phase t'tio
stabilizer nJfiuuanrn
'ld solvent fi hifi ;n q ;u rfr cr 6.r o r o d'o$ il iu condition6totllflTT mn t?llJ
dt q4.12 0{nil:vnoil tuor9)9r?outsclnTv 00nlJtTt
dtl col,rmn dlu solvent fir,:.rdu
4.13 solvent hiaruqn.61 oiq y( tUn:[u14 lT gradient)
ld mobile phase fi lirl#rfi qnrrruaruqnlu nr:oondu
t rd.t uv t{r fi {o d r riq yr r.r rn fi d r 1 #'hi ar ru r : n ld mobite
pnus. du'ld 1# *" gradient Ulanl< rtdrrj'lu-iauoofl0tn
baseline
- l'7/5'7 -
1lfufi'l fl'llfi91 nr:rrrl'1zu
5. noise spikes (fi peak 1u
baseline)
,lEl
?l
ffiNormal baseline
ttl,lllrllrl-JLill-
;;Air bubbles influid path
s. t fi l'losorntfltutardnA i' e/tqtq
rFlo0uylzulutljl tu Iu
rdulrsnr:'h{auo.1
dtsnunlu
qrio r.:
s.z fr r,''lo.r orn1fl1u pump
head
u ?fl 1 ci mobile pnur. ri.r o g 1 u : v d'l td u r riu u io ei r n'i rvvctY 4 v a o I d I
pump t?l Ufl 1JUn{ tu9l ltll'l 1.1 {?1Q ifl ?1 pump
tubing fiilrlr'flr pump t4Rllr io uioqor6'u 1#uryui#
' u cl I A t'[[uu 9t9t [14st: { u: orl6uuluri
degas mobile ptras" 'hi6 1# a"gut lilri
fitter froqjlut?e mobile phase dflil:n h{noq filter'hJ
vito'ttuavorpr]prudt'lil sonicatc 1u o N Nitric acid
nrud'rurir ; n9q rtovsrrrLd'ru methanol
tfiflnr::vrruuo.l rnobile phase 1l6lluoqj'lu pump head
'l# p.i*" p.r*p
rubing fi lr r i uli pu-p fr t u r or r#u r.ir qu tin n r{ n I u1u
r?o u rfi u hJ fi ov 1 # rnobile phase'lr n rdr h{nl fi uu hl ldo ,ia
tubing t14 }J VllJ lJ U lA tH lJ 1 v 6f lJ
s.: fior4nrerrfin1 oqjlu
detector llow cell
lirnertavolqfi;o backflush dctector flow ccli
5.4 n1:9rOdlu9tU10{
pump fi5Ofll:lSOlJfiOn t94 lrl I
tTll''l llo{ autoinjcctor Iu
[11]J'lVdlJ
1 da r u fi u r r n v 6 q r r u n Q rJ n : air rsi n v ry u^n o d r 1 d'il u iiu
dr a r u er^u'hj 1 ri a r rm q fi d r 1 #r n^ or il cy r r 1 #t fi u u rl d'n b4
1J O{ pump 14 f O autoinjcctor ilU fl fl 1-l n v ?.: 0 :'Iilfl 1
- 18157 -
1lfufi'l fl'llfi91 nr:rrrl'1zu
5. noise spikes (fi peak 1u
baseline)
,lEl
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ttl,lllrllrl-JLill-
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s. t fi l'losorntfltutardnA i' e/tqtq
rFlo0uylzulutljl tu Iu
rdulrsnr:'h{auo.1
dtsnunlu
qrio r.:
s.z fr r,''lo.r orn1fl1u pump
head
u ?fl 1 ci mobile pnur. ri.r o g 1 u : v d'l td u r riu u io ei r n'i rvvctY 4 v a o I d I
pump t?l Ufl 1JUn{ tu9l ltll'l 1.1 {?1Q ifl ?1 pump
tubing fiilrlr'flr pump t4Rllr io uioqor6'u 1#uryui#
' u cl I A t'[[uu 9t9t [14st: { u: orl6uuluri
degas mobile ptras" 'hi6 1# a"gut lilri
fitter froqjlut?e mobile phase dflil:n h{noq filter'hJ
vito'ttuavorpr]prudt'lil sonicatc 1u o N Nitric acid
nrud'rurir ; n9q rtovsrrrLd'ru methanol
tfiflnr::vrruuo.l rnobile phase 1l6lluoqj'lu pump head
'l# p.i*" p.r*p
rubing fi lr r i uli pu-p fr t u r or r#u r.ir qu tin n r{ n I u1u
r?o u rfi u hJ fi ov 1 # rnobile phase'lr n rdr h{nl fi uu hl ldo ,ia
tubing t14 }J VllJ lJ U lA tH lJ 1 v 6f lJ
s.: fior4nrerrfin1 oqjlu
detector llow cell
lirnertavolqfi;o backflush dctector flow ccli
5.4 n1:9rOdlu9tU10{
pump fi5Ofll:lSOlJfiOn t94 lrl I
tTll''l llo{ autoinjcctor Iu
[11]J'lVdlJ
1 da r u fi u r r n v 6 q r r u n Q rJ n : air rsi n v ry u^n o d r 1 d'il u iiu
dr a r u er^u'hj 1 ri a r rm q fi d r 1 #r n^ or il cy r r 1 #t fi u u rl d'n b4
1J O{ pump 14 f O autoinjcctor ilU fl fl 1-l n v ?.: 0 :'Iilfl 1
- 18157 -
d In td s d d 4gll:l{11 3 baseline lill5uu lfluilGIllrqlnu?luo't0ln detector
flcyur fl'tlil4 nr:rril'lcJ
1. baseline hi6uu lu- irn rarAnUilYYl lUtUl-J cycle
-tim9
Normal baseline
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Delective lamp
l
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,ll
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Operaling at highsens|lrvr1y
I detector rilhirndu:a a9/
[11]J:&Uy[?nl t?l dctcctor lamn tflfiU: lqUovrfiU baseline
o & q oqt , d 3[:UU1JU :VUY[?nl tilfl]:111[14 detcctor lamp IC{0U? lUnl-J
A a daqY I Asu 9r I o.1 detector I tn y y't'l : -t
lJ [Fr 0 : 11 tY [Tu n ?1]J u] ?n nu
se n s i t i v i t y. ba ck ground frn til r,^ht r [ [n y n : y t [d uh^ht
r r{l u piu
1.2 dctector lamp "itqer 9l:?0doU iamp energy ]eU1ilfiH#1 detector diagnosticsy L r o'a a - I ol q9 rAfi1 energy fl ln ?l[nilc'n[]J 0[1] trU nUl: ]J [S{lU [1'l lil n UU
o | | n 6 As)lamp'[nrj odtr'[:fiqrrr derecror u]iEfodttJt:orl:-l lamp
rnY j o I Ienergy l9ltlJOXJfl lnAU OUO.1
t.t finr:rjurfloufi
detector florv cell
ou4111n?tlJflvo'r9t9l.ru
i - de)1.3. I ttllUmn00il1-J 9l'lU union
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pt a ru rrh ri'u solvent d,, o ru,.r'l 6') sr r ru d''l u * a n t ;,--^q4isqy.atn,<Jqs,1.3.3 tUn:il?1tFlU mn tqU try mobile phasc UU9 []l)JU1 tl.t
v
flush d':odt: rudxJ5yil'j-ti THF rrnv rirlu dqr :'rd?u 50/50
,g)(dr ar : prar u o { THF nCI v d r fi dr rJ rT o p.rd}J rdr fiu solvent
d lqt 9)
?llfiU run t9l) 9l1u9l?U 100 % THF
t . :.+ t{r fr.r u u fl ey u r'l f n.rrh d'r u a r : n v o t u fi t r :'r'u*u
tn t9.l d1.4 :v1JU tvll,'i110{[n : 0 i
detector fiflgunr
detector "f r q or or^or ri ovr.t
AI1.5 d]UrryOil9rOiln?lJ
aeirlH:On1:rS0u9lo:eil?1{
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urtrJril ilrv dlJ
4rl9r 5 ? 0 6YOU fl
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1.6 nt:9roff "ru9ru109
detector 'hirnrurvau
1 da r u fi u r rn v sl^ o r r u fl e r-J n : afr r si n v v u^ n o d r 1 dr-J u riu
tir a r u o^ u hj 1 ri a r ru q fi d r 1 #rfi o fl ey r r 1 #r fi u u il d'n'l v,l u e.r
detector r[unnu ny1.t t: 1ylff rfiuqrJn: nfdu
t9/57 -
n1:tlfl',hdi v
1.7 lJd{5un?u fl iuiu'l6u
ioafl ?rri 0?lrrJ (RF) 1'r : o?.10 :
dt,r
Irun detector oofl 0rnoiln: nfdu ]qurqiltvodrl8rei a !< d,l rgt q | !,
lfi : 0 { tJ 0 ?'l }J }J 0 [a 0 : Iy'l il 1 I U I Fl t1.1 tU 0 Ifl U u
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flnu rv'rv{r (910 1.6)
1.7.2 Fr: ?0 doul# detector ttnefl ollfr?rsr ofo dlu aru
^ d u y o J" I
9tuttuut9ru?fl u o109lo{91qfl { tl1 lJ
I .7.3 or0 e{o r fltu detecto, hjl{luuinnrfi lrirfi q il flr il r
0tn RF r?o1d Faradav ,on. ]qg:oll detector
r.q fin-r:;?filLd reference I ryourriorrjduu r"f.r"nce electrode -'lttil
electrode (lQyllu
electrochemical detector
ril ruu/
t.s dsrir sensitivity ttnvad
.gain 1 O{ flO lJ1\ ?tn O : q.l
rnu til
td I dvqY9)[uouuntmfl{ [1.tu0un{
ril fi uu ar: av ntufi il : : 0 oti'lu reference electrod" 1lr riari
[tOv/1'l5OlilnUU frit
1.10 reference electrode
anil:n ([qr^'nv
electrochemical detector
r111UU)
oAam r n': t l.l dv o t 9l r 5o {9 fi r working electrode tir ri.r n u fl rg u r
'oY rioqjl#trlfi uu working electrode'Iuil
1.1 I working clectrode
l4dddfl lJ: fl 1.1 TOil : 0U191191
(tnvlrv electrochemical
detector rvirriu)
- 20/57 -
ilrgur fl'l[1.19r nr:rrfi'lu
2. baseline hi6ou lu- idr oRnUilYll[lJil cycle [lJT?{
J .^ 429 4.t?otdu6"l (?ut?lfl nut?t)
tbI
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IIl5t---t-+r
iiFCycling equpment
da2.1 ud{:ufl?ud6ufu'ltuoo
i^dn?ril O?vrrJ (RF) 1.r : o?.i1 :jqvytts
qrio r.z
vtao2.2 a{flloiuuniln'ru tuq{
Itr'lJo.1 dctector tlJ[1'l ]J',]vd]J
4^,i(intemal heater [lJ9 -lJ9lfi
rflll lc l)
d'wir i.rt"*ul heater'lrri
-?. baseline hiGou 1u
- id o I
CIn!ileillilU cyclc [UT?{.aarl,s
[?CI]UrU (UryrO tT? tili)
,l;tf,t.l
'iF,Normaltbseline
E
f,
ay n | ;3. I OruilnlJli 0 i tlJ9riltq{
y qy d y t1 q a 4 c9a r qil ?i 81J fl fl 111 r [1 9l o 0 u t].t ir i ]1 cl I u iu h I u .t rn q 1 u [ ?i
:. t. t &rornio{ lillilu anrilrr?qdoilfi fi oruu nfr nrfi
:. r.z odrl#ud{uaqd'uridrnio{ai
3.2 }J 3{{ : U n ?U Cf €U €UttU
4a4n?"rrJCI?l]€J (RF) 1.1 : O?.1 0 :jqv11 tY
qrio r.z
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Th.Normal basefine
4. I detector ti{hiradu: q{o r.r
^y n I i4.2 orufi fl tJl.t 0{ trun {vlqo qrio:.r
+.: finr:rJurfloulu
detector flow ccll
qdo t.:
4.4 reference electrode
anrj:n (lqy'lry
electrochemical detectorI
[?ltuu)
I9t10 l.l0
- 2115't -
q1Jfu111 flt [14 Fl nt:rrf1',h
4.5 working electrode
anr-J:nx?ofi:ougnfin
(tq Y{ f v electrochemicalv
detector rvirriu)
qrio t. r t
5. noise spikes (fi peak 1u
baseline)
€
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5.1 detector lamp "8t:"n q{o r.z
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n?rilCITuq (RF) Hio?io5jqvmlry
4{o r.z
5.3 :vuu'lvlfi rro.:rn ?uu
detector fifliurr
v9lcjo 1.4
4l.ll4ur1,/9ll :l {yl 4 retention time ltl ClUl{ilU A {fi :o-lilOn gton
ilruur fl1[1491 nt:rui'Izu
1 retention time lunr:fiFlYl
trqioyni.r trinxfi
l.I :vurlf,{'hirafiu: uio
frshi equilibrate 1l1.1tfl fi
^ dtI' r I
[]\ u t? n I I?l fl 1 U ]J : y n 0U Ft]{ cl lJ O{ : y U iltryU column tlO V
, + d ---- d I ddctector tJ fl ? I U td 0 U i I t0 U equilibrate ?1'l,l lft ]J O U'l { ty,i U.: yl O
louqtrnanrryfild rryu mobile phase nr:r{lfit detectora.dt9.l
t tnYSU9l detector [lJU gtU
q dd .. y q y v ytU fl : flJ yl run gradicnt 0 V 9l O { t14 [? n'l : y 1J 1J fl n iltt I d
| 'd d u I u I 14 I
equilibrate fl oumoyqnn?out{a?no lil
1 u n : tfi fi 1 d ion-pairing reasent o y 1 dnn r r rn y ri?l r ar
solvcnt'fl r u ru u dt lil fl 1 i equilibrate n o d'ruti rdu ld
dr:avalurdruflu s mvt druru 100 ml fi6'n:rnr:'lmn
1 ml/min rflu6'u
- 22/57 -
nt:rri{'h
1.2 n?llJfiilll0.l pump
A-!tfi:o0{11?vnt:uil
dt:ava1ufiqilnfi (oro
rfiqo rnfi ?'lo'r ornrfl fi $o
check valvc. plunger seal,
plunger "frqrt
fl : ? n d o U n I : li r { r u I o s pressure transducer tfi o ki 1 rtti'l
flow nr: fi n'lrlriu rflu nu d
Prime pump'lrrio r o alu
Fl : ? 0 fl O U n I : Tl 1 {'l U EJ O { pump ? 1 Yl 1 { 1 lJ [ 9l 11 { dO.l pump
414'1450 tlJ
drld rot,r",rt d:yrruiru 'l# a.nu, lnri
#r rfl u n r : run t tu u gradient o v rfl u a l rJ n st^ fi n'l r ru piu'hi
iA4nx fr fr I fr or rH E 0r n n ?r L il ff q fi rilfi uu hJ rff o dn : rei ? u u o{
,jmobilc nhase tilfi uuttilo r
oojie)qrfl il[9rtJ119n:t iyt 7 1o 5
ra4441.3 lJ:lJtA:nqaH:Ofl ?-UJ
9Ylr[1'N1U10nfl?Ouli]J1fl
a ll I o q9, q /h 'rnu til 111tl{floa}Ju tu
equilibrate
o pr rJ i l r sr : fi fi e H io a n n r r r ulil {u cr o r 6'r o d x d? u mobi le
, Y qY id I I
ohase d t "tS
solvent it fu a r t I t t:.t u o un?t o v atrLr : o fi e'ld
'hi rL r n n'i r 1 0% I o a il;ru r sr : vo.r'i r r 1 u rr o a''li ri r r si tir 1 dia
solvent fr fr n r t ru r r: I rJ'1 n n'i I 0 y d-t lJ I : rl fi et 1 d'l il il I n n'i r
to/o qJoril?lrn : ryo l'i r llu n oii'ru ri^g) h I i
1.4 0fuhfllJ140n tilfli11qg
. I q9,) AYq u ^ { .1s)n { q [u ?i Qil fl fl
-lT,r [ [?q a 0 il t 1.t fi J 11 n r il ry 1r 1 u.] [r-l9 r u |]l
1.4.1 ldcolumn heater fioruunfi 5 "c rrfroqq
^9ofll?f nruilo.trYvd
1.4.2 9l:?0dOU?l solvent zudlJttlnU9gt A a s /tneo jj
1.4.3 utuln: 0{1,1 : ofl 0nilu t? tud0tuTlilofiJilnu. y d 4 q 9u
flfl -11^l[t?nno]Jn{11 l.t:o tnnn]J qqIeuIuoIntfln ta
1.5 nl:zuCllJ solvent []J9 Fl : ? 0 d o u'i r il il H r tn^q o r fl n'l : r.t drJ solvent hi 6 r?wt io 1 ilL
lnufio standard il:yrurru : srfl lquidmobile phase fiY A tq a
p! dil [0.1 fi'l retention time Fl'111 t td9l $ ?1il 6U 1'l lln q 0 1n n] :
r'rdlr solvent hifio?l h{qrfiqrr:rs fr z 4s z.z
fl:?odoulqu run d'au column duluri
r . o. r tir hi ru u fl ry r r 6 n r r d Fr {'j'r fi n r : rJ u rdo u fi sr o d'ru rf
re^il oi r 1#dr.: u io nJ fi uu nr od'l rflr riu,
t . o.z dril iu H r &r n s rfi q rou r rd er.:'i r o'r o rfi pr o r n n r : fi
solvent'hi a r rL r : o p.r a ru r dr ri'u 1 d' m ?o fi n r : r-J u rtl o u fi
mobile phase r5o guard column ttio filter 1u:yul
4rAd1.6 ilnrrilu[lJOuvl
- 23t57 -
q116U141 flt[1491 nt:rrfi'h
2. retention time 'lunr:fiPtYl
tdl
rlnavfl:.:[ilaBu[[un{^ :: -x"[ulr
t rurnlLruH5onqa r
96rgtAslu taqtu l.t u {fl a oqt?a1
2.1 pump flow rate
,ilrilnuurrljR{
9r : ? 0 d o u fl r : pi'.r ni r dsr : r n r :'l fl o'u o I solvent h?fi n I r rL
UAd J[11 U
-]v dUn U 15 fl 1: m A dOU U U dl
^9J Vl I d2.2 aatr flN}j 0.1 tlJ Fl.lllqq
qrio t.+uhrdA
2.3 :vuuu.1 trJrdfru: 1.1:o
tis hi equilibrate ?11.1lfl fi
qdo r.r
drAd2.4 iln1:!u[lJ0uvr
flonilu
q{o t.o
u tA2.5 n OaIJU tdoxJdfily,l
(qcg rfi uq ar ar rTfi m rs tn fi )
laavd9l : ? 0 dou lJ 5 y dil t n1il u o s no n lJu
2.6 nll degas t'tio sparge
mobile phase'hitrrurvau
dr mobile ptrase'Iil degas rfo sparge dru helium ttdr
ri r'hJ equilibrate 5 ii u 1J -'l
u rj * fi rto.r : s iq o d r'l #n r : li r
heliurn sparge ri tb1dfl : rd?u lJ o i solvent nJ 6 uu'1 il
z.t flnr:rJurtlou'lu
mobile phase
rn€tlll mobile ptrase -'luri lqu'l#ar:rnfififiqarnrnd rray
solvcnt ryfiq HPLC rn:Fl
";lirnrrruavotel fitter riodlutrali5Q mobile phase loudr
filtcr 'irl sonicate 1r, u * *,rric acid ,-tudruri.t : n#.r trny
a1:Jd?U methanol
Flush trOY equilibrateSsUllr :.i
2.8 inlet filter Uo'l mobile
phase r?o filter'lu:vltu
qqei'u
Yr
er : ? 0 cf o il n r :'t r o yir : y r u'i r d'u fi o q 1 n o r o r{o s dr { 1 #arjol
fly0'19114:0lilnuu tilil
lirnrtrruavo-re filter fioqjluzuroruTT! mobile phase loudr
fitter 'hJ sonicate lu e N Nitric acid srrrud'rurir : nr:% rrnv
flrild1u methanol ororlosnJduu nit fioqjlu filter'Iuri<,Lao
2.9 lJnl: :?cillJ tll: uulJ er : ? 0 do u ntting v{{ il xr q dril u n r : ir dru fi q or 1 n fi 1 #n ry uqY t, u'rqsy' r al4t./u - rqV[l.tr[uu (:u?i0ut tl.rrluuu]n[nu til) f]'lunfl{:10q [14
n-J6uu fittins ttoy ferrule 'lrri
3. rctention time rilduutfluyr
rqrd4udafr't t1.{ }J ?'l fi { l1il nfl : w q9l
fl4fqrrAg,Jr[9t []J [ryfl ]yrfifi9l o.:q
io 9,'!
3.1 mobile pnure fildhi9t a 4u ' i
ftfl 9to{ 14 : ouo9r5-tc{?uvl
,", g o o ^dtlJQnno{cf 1145u?rnlT
v
lletdou#u
tn5uru mobile phase 1rru
3.2 pump flow rate
rilrilnuurrljRi
er : ? 0 rf o u fl I : ri'wit dsr : r n I :'lr a t o I solvent hifi n r r ru
UAA Jr14 u'r3 dil nil ?n nt: yl9 do l,J u u c]
- 24/57 -
ilcgrr d't[14E nr:rttl'lt
3.3 pump r4rsrufieilnel^o q 9v tn U I
111 lilOA: tflt: [140 tlJ
flflno.l
Ft:xrdoulauq9) - .j- tTfl:VU0fl9l?{1JU19l 5 ml ?1.110{:Udl:nvnlul]00fl
0]fl detector
- nrn mobile pnur. lorul# flow rate srtrLif;nqaoufil'ffrYd
ou].:uou 4 u'lyt
- rirursu flow rate fild'o?.r
#r lo* rate ltetfiri't{#u traql'ir pu,np dtlrufiqilns]^o?sOt/ d d[1.r8vren:'r{vr 7
^g) ur t i3.4 qiu?r QrJ14 O{ lilnsvl qrio t.a
u I ad3.5 n{nlqfu?rQtJ11
tnt9)column heater lilQnilo{
li oiq t cr s) 9)
riln uu otu1.t fl lJvr tu{'lu t?l fifl Fro iqqu
4^3.6 xulall:oTua10'1
-/tnt9)floailu tllflnflo{q
Aavqfl 5 ?1dOU1U1n"d : OTU n1 0 {fl AO}JU
3.7 fi stabilizer oqjluA
mobile phase 115O
stabilizer rrJd t,ru an ril
qY Atn r4u u dlT solvent n lilil?n0nUtdu
dtAd3.8 lJnl:ilur1JOUlr
floauu
q{o r.o
3.9 gradient delay volume
UlU0.1 fluidic system []J
I0flnoq
n: ?n dou'jrfi nr:nJduurnJ nsfi fluidic system riohj rryu
nt 5 tfi il q il n : nfzu aru ar: zu o.r fl I : run gradient rfl u d'u dr
vl u'j r fl n r : uJ d u u r ril o r o v r{o r ri r u I ru gradient delay
volume 'lril
- 25t51 -
flr:rifi s gr.fdr.rto{ peak fiorJnfi
flaur fl't[14E nr:rri1'lzu
t. 'hirJ:rng peat<
E
;;Normal pea!<s
7
No peaks
t.t ilqnrnrfl injector
niu fiqfiq vial, 'hifi vial
ro " ' j - a
oH tu9r rItl.'l u 1ilfl { il91,
l^utl4t1J:tJt9r:Ft?0U'l{ [U
tfiurvlo, nr:finhie) d d @
{luQfler0{, [1:Jqqqqnu t
rlu
ddd
9ln9n:t{il 8 t|ovgr]:lnn 9
1.2 mobile pttut"'hi'lun
ilio'iilnrlou
u I t4'1.3 n{nl detector [lJ
qndo.: rryu etlruurr
fl0il sensrtrr,lty, autozero
dt 9)
rlJU9tU
t0 rl,t 9/at a '
tu [9t[!Fl pump ?t5o pn-p finrJnfi qfinr:r.rfi I ilo z
Mobile phase'l u t lq fi 'lilrfi u{ v,l o'ldrst^rL mobile phase
inlet filter 1J0.1 mobile phase m6o filter lu:vt-luqfloi'ujiequat:'livt 4 1J0 2.8
v I qY 9/
9t: ? 0d0uI ravn{nr lil nfl n o {
r.+ lrild'rjfu '"rofidetector
9l'1 zero detector baseline
t.S ilcyurorfl detector
ISU detector flow cell,
power supply,'l'10:'lllff rdty
tljU9lU
9rnfl151.1vr i I {oo
arr1.6 nt: [90]J9l O:v?1',1]{
/detector fl UnOilYl?[9lO:
lnrtlJ[14 tJ lv d]J
atl9r : ? 0 d o il n 1 : lso lJ 9r o I 0.1 d] u diu fu 1 fil I tn v fl 1 T 9l { Fl'l
i ' qY 'ct o I A- - 2t- -u 0{fl l:rryoxJgro t1'r og [u9l1rlfi u {11Qnno.1
jq ttr1.7 mobile pttate fi1dhi
9)
frfl9r0.1
dott9l:UlJ mobile ohase [?1]J
yq49luaa<afi I 1,i 1J lJ fU 14
-1
U 1\ : 0 il n U n ?',I ru 9l U llt tJ zu U O',i 0 tfl 91 0 ] fl fl 1 T
U'anns fl0u1Jo{gr?0u'l{
otA1 .8 9r ?OU'1.1[do]Jdnlv'l 9r : ? 0 d0u dn I y't lJ o { 9t ?ou1.1
addutJ?1?U d0 u ?r il1: ra 5 uil 9r ?o u'1.1 u u
dvrql[9]:urua?out.l n4]J
- 26t57 -
04
uful.t1 dr[14E nr:r[fi'hddrA
2. peak IJlJU lFl[nUn?lil filFl 11 ?.1
(qiU[f,U sensrtrvrty)
.ll:l | , I ll:l l,l ll ilt iltt ti lll_ JUr.,]r-_JL__J r
;;Normal peaks
I>lcl6l-l
l-- lu= -\- -;;;
Smaller than expected peaks
ra dd h I
2.1 ilTrJ19r:ilqq t:J
9)
61fl9tO.1
ld 16 dd[!nuuu:]J19r:mq9l
2.21Ul911O.l sample
loop 1u injector hi9)
Qnfl0'1
el : ? 0 dou sample toop fi 1d o r od'osn-J fi uu 1r ritfJ uj
1JU t9l?][1.1:JtS fllJ
z.: d.rrir detector'Iil9/r
c|flgro{ tyu fl?]}JUl?
gldU sensitivity, autozero
dt 9)
luugru
u I qy 9J
9r:?r fl ou[[Rv9t ifi't [14OnR0.:
h 'tn9/ r- )2.4 LU t9l1j5U zeroYl
detector
91.1 zero dctector baseline
jtl
2.5 n'r5 rToufl 0 : v 1'l'11.1
rY4i
n : ? t fl o u n'l t t1 0 ! n o 1 0 i fl -i u d frj €u I iu | [a s n'] t fr i fl Ioo
uaddetector fl iln O!?'l?[FlO th'tu[14 ]J tV d:J
4 r 619/ t6l o I a I10{nt:[YolJ9lO tfi Ou lU9nt tfi USnfl nnontv
drard2.6 ilnr:ilu[lJou11
detector flor.r, cell
firn'lrruayorn6'{fii - dq
2.6. I Itl'lU?lfi0OlJUFl?U union
z.o.z lun:tfid,uu *n leul# mobile ptlur" drflu
frn|rdof 1# nusn rftuti rrrild'?u methanol (dr
methanol d-tlJt:CIp.tdurcirfi'u solvent d,uu *n ld'l srrru.yy
Y o49r?uutofln:{
a 4i * d u Iz.o.: lun:tfrfitnu run lnuld mobile ptrase vfror'hifirTr
1# nurn d?uar: 2oru : vH'irn THF rrnydr'ludsr: rriru
5 0/5 0 (dr dr : r.r dil,u o.t THF r rn y lir dar ru'r, o ^
au,,?l-ihftu solvent lttfiu run tsl) srrrud'ru 100 % THF
z.o.+ tirtirvrliliyrr t# nusn druar:ovnrufirr: rdu
2.7 detector lamp "irqor et:?0do1l lamp energy leulilfiil#r detector
diagnostics dt "n"rgy
ri r n'j r rn ar qirfi o tfi u u riu r?ru 1 #
lru h{nlfiuu tamo lrri odrsl:fintru detector ur{4-Y tv , rnvA a I vu?1 od]ul5 61 lJ 5 u lamp energy l9t tlJ olJ n'1aq u oua {
-27t51-
3. peak fi oonturixrr:nro{1fl :
rut lnttn: ru fi n'nusuv broad
. Time
Normalpeaks
t^ d4 43.1 1j5il191:V]AAH:O
YYvlFr'lltJ[lJ tJ 1U 1J 0.19t? 0 U1.1
^h';qtudillnlnu til vn t?tn0Rilu
'hi equilibrate
:l
a n rJ? ru r n : fi fi e H f o a q n r r ru rc?il {u I o.r pi'r o d r.r d'l o
. 9) ctt) - -ia y I
moblle phase 01 [T solvent yltJ9l?11J[[:.lUOun?l 0V
a r il r : cr fi q'l d'l :.i ru r n n'i r 1 0% 1, o.i il;il rsr : so l'i r s 1 uu d tY 19J j4
n0n3JU It9]fi'l tS solvent ?l]Jfi11]Jt[:.1U-1flfl?t 0y
a t r t : o fi sr'16'1:.i u I n n'ir 1 % I o{ il?ilrfl : roldt lufl0nuu
Fl:?0dOU tubing. fitting [tOv fcm,le fild
,l€lbl r i?l il,I l\ /\I-.--JVLJ\ J LJ \--
;;Broad peaks
(earlv eluters only)
flr:rrri'h
Z.S ilryrtorfl injectorr 4 a t4 rdtlil A9zuq vral, [uU vial
'.1 o tio)aou tu E|lttl.t u.1il fl {aq.
,lt-,rrrUrr,,,trf -,4Ahl
tylu.l?,1o, nltaq til
qnd'or, rfi rufi or qeroiu riJ u
Ftu
j;!qmmt:1.1?t 8 trCI3Fl't5-t.11'l 9
aot[0001.19t?ou].1
@r4d2.9 a'l o ut.1 lJ fi ?'t lJ ?1 1-l 9l
tfl1l [11u d o q r d d
n9t o9t :ltt ? [u n']5 FlFlFt ?0u].11JO.ltUilAAU
do td3.2 tubing 11 tSlJUUlFl
Y I d q l4r[duzu]fluufl alnnlu tu t].J
n'^QflFtO'1 1.1:Ofl',l:9191
ln rd a o 9,/
tubing [ilFr ?'t:o IT firting
[t0Y femrle'hirurLryaru. -.:
3.3 tun1: 09l9tuu1.1d?u yl
^^=^in-line filter 1150 guard
4udAcolumn H:OflOOIJU 14:0
trr fir n o
9) u i ou 'drqooi'ufi tubing l#nifi uulrri9uaAAv?fi'lO9l9lUll in-line filter ttiO guard column U5OgrOn'Uuq yvh{o r wirn rril dy o rqilf onJ 6uu 1r ri
:.+ fleyurorfl injeuorrl,Jad4
ruu ?'rn1:?ryuH:ou9laoddv4flqnu [uilq9qflauH:0
"frqo injector port Qord'udl 9)[uuau
iJi9rmFn5]{m 8 ttnyfll:1.111 9
3.5 tJUlAlO{ sample
loop lu injector'hi
qnno{
el :'l 0 dou sample toop fi 1# or opfo.rnJd uu 1r rirfl u
uu'191?1tl{}Jt&du
- 28t5'7
1J 6U141 dr[1.14 ntsr[d'h
g.O ri'wir detector time
constant hlqnd'os
qrdltainl [?tu
o 4q4. peak Yl{H:JFlilOflUftJV broad
o
;#Normal Peaks
9
T'mBroad peaks (all peaks)
drArd4. I ufil:lJU[1J 0U11 guard
Aqdcolumn 14:0fl0OuU
9vdACI1{nOAilUillO guard column
tdvd4IUO UUfl OnilU 11 : O guard column
- aA4.2 nOnilUld0lJdnlvl
n rfl qru tAuil : v fr n fr'n rrnr
Aarrclfi50lnqs0.1?1.: [u
ftodlJu
er : ? I do u il : v fr il fr n r vr cr o': fl o d'L f flr ei r n'i r rn a ryi
q q Y ti - l6t r
u01J:u tl4tllnuun ontuu t14:J
4.3 guard columnj
ldolJdfl'r1\
fi o ri'r o tj r.r J q u 1 il 6'o s fi guard column d,r'ld' peut rJ n 6
rtdn {'jriliyl4 ttfi q 0'l n guard column 1#nJfi ou guura
colr,-n lrriAq gt
4.4 solvent il tryRYAlU
9r?out{rufl?1uil:.1I
il1flfl?1 mobile phase
nuotuti'?odt ld':u mobile phase rr?o1# solr ent tifi
n ? I u ! | : n #o o n'i I mobile phase m ? o fi o pi'r o eir.:'l ut4 A 9.)
lJ: tJ 191:l]U 0UA.1
A^4.5 1U-r9r145oSUqUOn
- /t0' 9)
flonlJu ttJ0nfl0{q
4aud9l: ?0 floulJu19t14: OTU fllJOnflOAlJU
^9)4.6 0€lrllRtJUOnqU
td l4 |rllauu [1J
qs) A ^ a dlT column heater [n 0n?ilfl]JOtU 1'lR]Jfl OnilUqq{
ryntd4.7 :yUUU{ t}JldctU:
4 ul0 t
1'l:OUi Iu equilibrate 111.1
tnlJ
rfi ru n o r 1 #ar u rJ : y n s r qi r.r cr 1r o { : v u u tsu column
It0u detector finrrrurAfiUT [[OU equilibrate ?1'l{lflfiI d s iqy | -
oul {[1^lu{1,{o t9luq Otndnl]y yt tT [Y1] mobi le ohaseq'I
ad9)nl:fl{nl detector [[nySUft detector []JU9lU
1 u n : tfi fi run gradient o y rio r 1 #n a r : v u u n d'rL r{r 4
'jdutvtleouilibrate noum 0vaqnxout i9l?gto [lJ.
lu n : tfi fi 1 d ion-pairing reagent o v'ldtta r t to v rJ?ru r iu
solvent'iiu ru u ds lu fl 1 T equilibrate n o d'lL tf niu ld
dr:avnrur{ru{u s mlr,l druru 100 ml fidsr:rnr:'lrn
I ml/min rflueiudaad
4.8 n?tilt:? tun]:v,turlf4r9)tilflfl9ro{
tv d q a du : un?trut: ? tu n]:T'tu1.,,1
- 29t57 -
o4
U6U?,I I dlrlJE nr:Itfi'lt
5. peak fi d'nu arv rr fioutfl uY u 4 dl4 t
peak SOllflU 14:0}J [1] R
Itl?l I I Ial t I ll i\?l lrI 11 llI t1 t il lt ill- JILJL'Jr-_J t.--
;;NormalPeaks
I:l I r ,-l_{-L_l\iL
s. t fi nr: qorri'ulr.r ritufi
guard column 14t0
ftoouu
yuda9ltdcll0lqAnUil tubing t11[lJRUu t14tJ
iluiaa@J#rqqd'ufi in-line filtcr u?o guard column rSonon:1ilq 9)yr il n rn r I f l 11 1r fl v o ra11 io nl 6 ou 1u ri
dtatd5.2 uil 1: 1 Jutil ou?] guard
dvJcolumn 1'15On0AilU
iludaRlin0R:JUfi I 0 suard column
ldv6a[lJOUUfl OnilUH : O suard column
- tA5.3 flonilutflolJflnlvi
01nnr: rnqryo.r'ir.:1u
nonruu
el : ? 0 d o u il r y fr m fi n r rnr r o.r nr o d'rL rf tlr qi r n'i r r n ar qi
- qY rA - /clu0tJ: 1J tl.trllnuufi 0 nlJu tl.lu
5.4 guard columnj
[doildn]Tr
fiqei'rodr{lerukielosfi guard colum" drld peak rlnd
[tdq{'jt il iU t4 I tn^q o I n guard column 1#nJ d ou guoro
"olumn'lrriDouble/shoulder Peal<s
t^dda5^5 ]J:lJ1$15?]AqH:O
n rr il r{il slu t ot d'r o cirr
unrfiuhJ firldnod'rurf
'1il equilibrarc
o q il3ilrn : fi fi n il ?o a q n rr il ulrL du uor o{r o dr s dru
mobile phur" 6't1# solvent fifinlltr:l#oun'jr os
nrtl r : cr fi q'l d'kir r n n'ir I 0% I o{ il;ursr : ryo.r'ir s luv J ,e) qy - ia
n0nlJU [[9lCI] tS solvcnt ?lXJfl']'lut[:{}J1flfl?l 0v
d1il rT ofi a'l6"irir rnn'ir I % t o Irj?l rsr: vo I i rI lu
floailu
6. peak'hjaruilrfl : drur#r
(fronting peak)
ffiNonnal Peaks
l
TfrFronting peaks
r^iaa6.1 1J5tu19r5TQ911450
9Yulfi?lu [1]J1JUlJ 0 iFl? 0U]'1
xr r n rn^u'lil d r1 #n o d'ru #uhi
equilibrate
o e il?xr r fl : fi fi pr v ?o n n rr r r ru r{rL du t o I pi'r o dr I dr u
mobile phur. d'i1# solvent fifinrtrLtt:srioan'jr tv
dril r : 0 fi q'i6'1riru r n n'ir 1 0% tu o{ il;rL r n : vo t'ir I 1 uu d tY qY - ja
n0nLU [[n0] tS solvent ?1ilfl?1ilU:.1]J1flfl11 0U
d r il r : 0 fr q'l d''hi rL r n n'i r I %'u o r il3il r n : rio.r'i r.r 1 u
noailu
6.2 solvent fildnvnru
pi'rodttfin r"rilrr:.1
Ulnn'j'1mobile phase
I .. a qY . ddAyAlSgl?0U1{9t?U mobile phasc 145O [U solvent YIU
n ? I xJ [ [: { #o u n'i I mobile phase r io fi n pi'r o dt'r 1 u
rA Ac)lJ:lJtgl5llUOUn i
dtat d6.3 xJnl5 lutilo1.J11 guard
Aulcolumn 1'l:0fl0ClJU
dr.rnodlLrjuio guard column
fdud4[il nuun oniluH 1o suard colurnn
- tA6.4 n0nlJuldOildnlr'
ar16l01nn15lfl9tSO{?1'l IU
noalJu
19 o I Ja : ? 0 do lt lJ : y dm 5 fl 'l
1^'t I o { fl o n }J u fi 1 gn fl ? 1 [n fu c?l
- qY ri - Jq I
uolJSlJ tl4rilnuuFronilu t14 xJ
- 30ls'7 -
ilryrr dlrHq nr:rri1'ln
6.5 guard columnj
[doudRtl.i'l
s'qeTrodrllqulileio{fi guard column tirld peak rJnfi
rldq i'jtil iUil rtfr n o t n guard column 1 #rrJ d u u guuro
"olu-n'lril7. peak lilaruursr:druvd'{
(tailing pcak)
lr?l I , I IIt [ [ ll I<l lr |l ll 11I |lt il il nI IUUL--JLJ T-
;;Normat Peaks
lr?l Lirt3l lill llot lil |l il /ll_-]Uuu!_J L-
;;Fronting peaks
dl4t47. 1 }Jn]:ilutilout't guard
4udcolumn 14:OflOnlJU
YvdAA1{nOnilU ?1 I O guard column
ldadAlilOUU nOO ]J 1-111 T 0 suard column
- dA7.2 fl00ilu[d0u c{nlv'l
ar16l0'lnnt:tnqs0.11l.1tu
n0nlJu
I a a u ?gt i r JFl : ? 0 dOU U : y dnI n IV'l I On f l OA il U n Ifl I n ?Itn ilcl1
- qS) tA u dqU0u: U t1'l[1J RUU rlOnilU Il'J lJ
7.3 guard column
AtdolJdnlil
fi pr ri'r o dr I I q u 1 il elo { fi g*ard column tir'lei' peat r-l n fi
t I da {'j I il [U t4 I lfl'Fr 0 I fl guard column 1 #nJ fi u u gr.uro
column'luri
f .+ ilErirorfl injectort a L a aze
lYu ?]n?:?ryilr:ou9lvddqa
nonu r1jilqqqnnuH:o
^9^-^ . ^^^-T l!91 rnlcctor port QFl9llJ
<t 9)
[uu9]u
911191-t:1{yl 8 tlnyq151.1yl 9
Z.S 6'snir detector time
t4r9)constant []JQnflO.1
6'qnirlyri
8. uogr peak [[UU 'hitruaru
l
Normal peaks
ilma
Tailing peaks
a.t d'lrir detector 'lil9)l
fiflgro{ [ryu fl']'luul?
fl0U sensrtrvrty. autozero
4e)tljU9rU
vtcty9)9t5?0 flOullnyn{fl I tH0nfl 0 i
8.2 recorder input voltage
1ilqnd'os
rJir rirn rrlsir.r frn #lylft r1n ri
raAda8.3 lJ:lJ'rgr:ilqqil:O
9tYuln?1il['uutul0nn?oul{
aVtloct/udru]nlflu [u 111n4flOnilulntIu equilibrate
tA'joug9J9)ut9)n q il : ru r 9l : 1] a q il : o a q n ? I ]J 11 lJ u u u 0 { 9l ? 0 u I n n ? u
- t q t - io Y I
mobile ohase fll [S solvent yl]Jfl?1]J[[:.1X]OUflll 0Y
dril r : 0 fi q'ld'hiru r n n'ir I 0% I o i il?ru rsr : ryo rir luv d tY etl) ia t
nOnruU [[A0l [5 so]vent ll]Jn?lU[[:.llJlnn?] 0U
a r il r : n fi q'l dki ru r n n'i r I % I o { il;ru r sr : rio.:'ix 1 u
nonlJu
- 31t57 -
ilqur d,UlJE nr:trfi'ln
9. pcak nn'l#r (negative peak)
[fl peak
-Iido
Negadve peaks (all Peaks)
e.t kiarui{'qiyreu-,1,a/
ntou gr o1 0 in0lJ?\ ?[9] 0:v9
naun']u
o I u 4i t a lqY II a a r u iY q! 6U'r ru rqt 0 il fl 0 n 0 il v{ ? rel 0 : t }'l Q f,i el o.l
yluld
9.2 glifll polarity Yl
a^ddetector H 5 OnOilY{ ?1910:
'hirururvaru
d'wir polaritv lrri
10. peak n#lrir (negative peak)
U'1.1 peak
t,?l | , I il!t I I il irrl lr ifl lllJuLr-L_JL-Jt-
;;Normal Peaks
rl,t | , I I:t l l il tl?l ililil tl
l-uL-L,uJ'*;;
Negadve Peaks (one or more)
)^ il0.l rilu peak frtnrrzuu
'lu:vriu'uol ion-pair
separation
v19/RYO-lUgl ?OU1{9t?U mobile phase
tu 1.i cl 1t . -.-a dtj:t-ttilnuu condition [1]fll5 run [14 peak Yuglu
d'llJ15 ilItUfl 0Ofl 0lfl peak ttO.t analyte
to.z 1ud'rodr{f,
dr5il:vnoilfifinir nr
rioun'jr mobile phasc
a : ? 0 d o u'jr pcak n d'u fi'r ri'u rL r t r n pi"l o dx v i oa r :
rJurflou'lu solvent
- tilrurornrirodr{l#ilfurilfi uu condition lunr:
nlnv
- tltlro tnar: rlu rtl ou1il solvcnt 1#rrj fi uu solvent
luri
10.3 mobile phase
qflndutra{'ldxrrn
atynYAlU9ll0U'1.:91 ?U mobile phase
ilfunJfiuu condition 'lunr: ,un 1# peak vfinfi
f{l}J1: filtufl OOf,i0lfl pcak 1O$ analyte
n J fi u u nr r r ru u r r nr du fi ldl dfi ni r q.r n'j r uv-cut off 'u 0.1
v
mobile phase tiu
trjd uuryfi qto{ mobile phase
10.4 autoinjector qfi
ornrgr{r'hJ'}u:vutl
jie)9lflgn:t{il 9 u0 5
- 32t57 -
Av4Ylaultz)9t I 5 t.r fr 6 tJan I {t(i ru q iun I il fl t o n I no I ulj : lJ I cu lilQ n n 0 {
flqxr flrtfiq nr:Irfi'h
1. 5311. peak zuOS analyte fie r.r larirlu:uuil{'anl:
dorynhignd'o.r
L L I a : ? 0 d o l n r : l a ri r r r a v rJ :-u r t fi 1 #q n rlo.r d'.r fi
- sample table
- calibration table
- refcrcnce peak
Ju- peak window t6'{ 1 idou riu ilioqir s n {r.r tfi u hJ)
- peak threshold
- retention timev,
1.1.2 tln#u run standard luri qzuafild'
1.2 retention time
rjlTlJOUU[[Un{
9tmgr't:'lnil 4
z.'hirJ:rnn peat< z. r'lcirirlu :vuud'qnr:
riorynhiqnrlor
q{o t.t
2.2 retention time
,it'lilauurlila i
9l?19|t:1{vl 4
:. rJ:rnn p"'L fikinr:fioss-tU [fltru1 t9ltlfl:u (ghost
peak)
:.t finr:ilurtJoulu
mobile phase
rniut mobile phase lrri lpruldar:rnfififiqflinrv{fi rrnv
solvent rfirr HPLC rn:Fl
lirnrrtavoter filter fioqjlutrnIIIQ mobile phase lotuti'l
filter'hl sonicate lu o N Nitric acid srrrud'lurir : n9.: rrnv
qrf rudlu methanol
Flush t[03 equilibrateT Ululu rj
:.2 d'rotixtfiorLanrnvadrarcl
H:Oilnl:ilu[lJOU [U
rdul: vfi ?l{nl: tglT u}J9l?0u].:
a5?0c{ou]nuflt:fin standard #rhjnl ghost peak Itda{'j-1
q r q9,, tr'q s.iltuH]tfl9r0tfl9r?ouli t14[tfl [u [0u
- 9t: ?0fl ouflnlnro{91'?od1{Aa4vrJ- vr?udou?5fl 1:[9]:uugl?ou].luu
durot- [Fr:u]J9r?0uln lil]J
3.3 ar:rJ:vnou'luutiotYA
9r ?o u-t{ nq q fl 0 u 1'l u 1 [v{.1
0&tlfl elute OOfllJl
rfi ru n n r 1 u n r T run ri"t o d r': rfi o 1 #a r : rJ : v n o u Q fl elute
oonil1l.til9r
Y a dY - dd .-C_ dn1{n o a ru u 9t ?u solvent ?1il n ?'l}J [ [: { }J1 nt?{ oryv fl 1 : yl
c{-r rJ r : 6r n e.fi'u o d 1 u n o d'lr rf 1 du r u o o n 1 u : v fi 'j'1.1 fl 'l 5 runq{
ydd3.4 :vUUO'l{[Xilq9l
flflun9r
qfinr:xfi q {o a.:
- 33157 -
ilcgrr fllr1.1E nr:rrd'lci
:.s nr:dx sample loop
'hirfro.rrnro
9,,yrdrnli sample loop 0t'lU mobile phase TY11?1{fl1:qnt[9]Ov,,
Fl5.:
:.0 rfirufiq'lildvorerc'vd a itry[uilaqTflvo'r9l
dt.: injection port
:.2 finr:rJuttJoufi
injector
dx inlector [tOu sample loop
rr]duu seal [[03 filter
drArd3.8 ilnr?ilurlj0u11
floatJu
9)v(nr{flon3Ju
,) ^, lrrlnuunonruu
d to
3.9 lJ stabilizer 0Q t1J
4mobile phase 145O
,istabilizer lljouu dn111
o e, jm 'o- v d A I lv'[d solvent fi 'trifi igronu ldu ryi010 91 0.1u: u condition'lu
nr: run'Irri
+. r1:rnn p"ut riounitti
nrqfiqlulnirltlelrn:tL
vt4:1 I n?0U1{[do]Jdnl]^l a{o r.:
+.2 qtgtfio resolution 1r er d o il il : v fi u fr n r vl t o { o o d'u f t{r #o un'i r rn u qiu o rL fua9) rA u dl14t1lRUUflOnilXl
jo9u,4.3 mobile phasc 11lS tll
9)
c|flFro.1
rsr?urL mobile phase truyr
Yo4dY^44fi '111 il | [U 1'l 1 U l','| 5 0 :J fl il n 1 ] ]J I U t]\ ]J I U O 1 o tfl 9l 0 I fl fl I :
n navfloulJo{a?ou'1.1
5. q€U[dU accuracy 5.1 nl:ri1u?6tt peak
height r$o peak area
flflp{o.l
14rIU
5. 1 . I sr : ? 0 do u n r : 1 ri ni r r tn v rJ fu t r r11 #q n o{o I d't fi
- sample amount
- scale factor
- intemal standard amount
- retention time
J o , i',rv5.1.2 0lnUU run standard [14]J qt{nm l9l
o ai -s. 1 . 3'[u n : tfr fr : v ti u o'q n r : rio q n : r u { 1 u t't a componcnt
amount finilniq l#nJ:^uurfiuu response tos peak rilnir
1JO{ standard tO.l
- tlrr.r u'j r response q n r{0.: [ [e{q {'j I il iU 1'l'}]J I 0 I n
quantitation
- t{rvr u'ir rcsponse hi qn do.r r tdq{'j1il iut't til t0 -Ifl
integration u6o chromatography
- 34t57 -
lJiu141 drrl4E nr:rrii'lrut4
5.2 9l?OU]{[dO]Jdn]ilv
4dtdt q
14501JflT:1JU[110U [11
r4:v11 ?1{fl 'l: tgr : u}J9r?oul{
q{o:.2
5.3 lJnt::s[14U1JO{
ca -la F11.1
d o r o ^YdSeal ltCIYlfl U9l?oU'l{ tuqruilQilqluu
5.4 nr:rn;urud'rodrshl
Qflelo{
dorrYYc3lll?UdoUnt: [9r : U]Jg]?OU]{ ITU fi2lrJ[UulJu fl ]: n : 0.1 [1JU
glu
5.s nr:tn6uil intemal
standard hiqnd'o'r
d q , i.lY ' ; - ali lU dounl: [9r : UtJ 091 : r d?U Tt tT ITU U lil U nllnvfl '.l: [0 0
dty0'1.1 tljilau
[nEuil intemal standard 1uil
s.o ilrgrrortlnrsfi q ('luoiq Y
nlOll'l lT external
standard)
drld nxeo-toop manual injector 1#1aar:ovotu'fi'lu?u 3
tvirzu o{ il?r: I Fr 5 loop uii'hJlu toop riou d o g d'{ fi el
dtld partiat-loop manual injector o v plol fi q ar : 1u rJ?ru r sr :
fi#oun'jr 50% 1Jo{il;}J1Fri sample loop
v qr)fl1tT syring-type mauual injcctor
^61 49d;- [llftufl [ufl tTqanyno{iln?tiln{l]
- ldturqto{ loop qnd'0.:6lt a d 4 I- [Tyu9ru0{t1ilq90fl ao.1
dd- l1ilqflfly0191ln ki' <- loading port llJS?SlJ
y q9fi1 [T autoinjector
qy 9)- lry1U191110{ loop Qfi9loiQ9 a d d I- lsryuqloi[tilqqQnnorh rd q d d- lililorn'rfl tu[1]Jq9l
4v I I d- vial lJFl?OUl.lOU[l.''lU.lltO{l4 rd "i .9- []Jufl1::?ryil
hi'jr o v tdt ru il manual u?o autoinj ector ov d'o s vi i'Ifi
rduyrxnr:'l?to equilibrate Jq u
- ?t.t manual injector lilusirrrurj{fifl
- purge autoinjector
- 3s/5',7 -
1J tU 1.1I fl'l[1.jq n'l:rrfih
6. qqtdU precision 6.1 n]:d]u?fl,l peak
height lt6o peak u."u 'hj
qnr{or
q{o s.r
o.z flqmrornnr:fiq (luoiq Yn:filll [Y extemal
standard)
q{o s.o
O.: flcymrorfl injector [sl.J
Ai'-<a4at'lo ?: ?ryufi: ou9rgr9tflu
ddvAo[1LA9qqflUH5 OY']:"91
-dt9)injector port Q0au tUU9lU
91119n1'1.111 8 t[AyAl:t{11 9
utA6.4 9| ?0 u]'l !d0 lJ f{n lv'l
4draro
o{o :.24dtAtcl
?i:0iln1:iluru0u lurdul
: v l.t ?1.1fl1: tgt: ulJ9l?OU'1{
Q?]Fn:"r{11 4 [rAVn]:l{?] 5o.s ln : rLrlsr rrns:Jfr fl cuil'l
lS1-! retention time
ri l 4 tlrilauulruni il:oau:lit^
peak [.l9lUfl9l
6.6 response fild'orn
detector dFlO.1
A or crl)fll:lnil 7 il€ufilnr:luir1l{ pump
o4
1ifu14'1 er1r14E n1:rrfl'lu
1. pump hidxru
nru[tnU front panel
lixrurryuri'u
s .i-[9ruvtTl9l
l,t r
lru
ln rl/tt4 tu h I
1.1 trJ torduuilnn trl 9l : ? 0 dou n1: rfi uu ar8'ly,l
lr rd Vt 19, d1.2 trJrJfl:v[[d [1^lTllvl
I d ru 14 r
rgt1lduuilon tyl
sr : ? 0 d o 1r reir rfi u u il d'n'l il 1 or o ri r q rl n : srfl v'l fi r fi r nj 1 o'j rcrll tiy d A gt d v"[ {s r u'[ d u r rfr I u fr rei r rfi u u rJ a''n 1 v'l fi tir q rJ n : afl il fi r hi
fi r r r u r rae q'i r refi r fi u u rJ d'n lvl fi il iu u r hi'nj fr u u rd'r rfi u L
rJnhlr,^'l1uri
1.3 fuse 1]9 n]fiuu nrse'[mri
- 36157 -
ilryrr dt[1.19t nr:rrfi'Izu
2. solvent hilrn 2.1 fuse 1l9l njfiuu fus" tnri
2.2pump hjrfrorLsioriu
pump controller
dltuA : ? 0 dOU nl : [5O]J Fl 0 : y 14 ?1.1 pump f]U pump controllcr
I y a9lt to v fl 't : 9l,: 9nt[1.1 u.1 1J o.: IEEE-48 B (6 1 tu IEEE-48 8)
qadA'il 9l d?1'l T pump [ lCI U pump controller [ [6] U 61 0O dl UISOIJF| O
99
oon olnfutflqcfiytli pn-p 6nnfl tit p.rnp ntilt;fi
ri r I r u kilq u hido.: ld pump control ter t r fl et {'j 1 pump
controller fi il cy u r ( #r ru o o e o r u rfi o ru si o 1 u t ru v fi q rJ n : ofIu al a d r.
il{c{o.1lil9td??tsog)
2.3 flSn'l low pressure
n9/ |
limit 1J0.1 pump tX({n?1
- iqYfl'tluflu11til{1u
6'lnir to* pressure lirnit tol p"*p 1nill#qnrlo.r (ovelol
J , - Aqgt y I o qynlfl ?'lfi?'llJ9l Ul1 tU{lu 01q in?10 U ?11 ll4 pump nqnl:
111{lU )
z.+ #.rdfl : rnr:'hi oto.tdrl
pump [UUfluU
14
tlIUOfl;tn1: lltA
2.5 pressure transducer 'Iil
'6r . ' id 40 u tu 9nill.i u{ }'llu u fl u uqq
1.15 0vr:91
il ilt prerrrr.e transduccr 1#ril u qu ri'lu I €u v fi dn : r nr :tndr?tHArllUflUUq
cioilHioril6uu Pressure transducer
2.6 lilld'prime iltt prlme ilru'lrri
2.7 draw-off valve 1'10?1J
4o,"sH:O:?ry}J
14tl1l draw-off valve 1ri'rnju
flJfiuu seal1o.1 draw-off val,r" htri
2.8 solvent'Iu pump head
'hi a r ru i : n p.r a ru uir riu 1 d'
et : ? 0 d o il'i I solvent dl lJ'l : tr zu d lJ r{rrYu 1 6'u io hiA q9i , d I u vt9)
lnOfl tT solvent yldltJl:ntiflil[11flU t9l
gtApurge pump 9l?U solvent 11tl1lJledlJ
2.9 check valve dtu inlet
l4io outlet anil:nrio
dr.rrufiqilnfi
et : ? 0 dou'jtfi fl cy u rfi inlet uio outlet 0 tfl 1il-l dr.: li rn r rrL
ariolfl e o19rl.t : otil a uu t1.1 lJ
2.10 plunger seal "f rqot ififi solvent frdruoonvrrldrurfrl pump head Niorfiea9.r'
9l v fl O U [fl n 0 : O U 61 11'ln9l I U t4 0.1 pump hcad [ ldn i ?1 piungerqv rj
seal'it!pt .tfnjduu
seat lrri
-37t57 -
1lfu111 fr][1491 nr:rui'h
z.tt fiornrg'lu pu*p
head (tqy'ttv pump 9l?1lJ
ri'uq.r;
cJ r Fr 1 d mobile pnur. ri'.r o qj1 u : v d'il rd u rrYu u ?o pi r n'jr99cl I 4 oO d I
pump t 11 U fl 1J U fi { tU Fl -}
[ [?'l u.1 11 Q.l fi ?1 pump
tubing fimr.:uh pump 14o?u ro uioqnri'u 1#rr1ul#trrjuu c1 Y a ri oq'fl 1f n : r m To ujd uu"lr ri
degas mobile phase 1il6 1# aegus 'luri
filter fioqjlutler mobile phase dnil:n 1#ooo filter'lrldt
fi?'ltJcjvola]nudrlil sonicate lu O N Nitric acid qtile{?uvv
rii : rrir [tayfltilel?u methanol
ln^nfll:5vtHUllo'l mobile phase 1J€tJUoqjlu pu*p head 1#
prime pump
Aydyr.?--q9)tubing 11 11 1 n t1J 1 pump lJlllJ1fl tdU ft 1qil Ufl A 1 { n 1 U tU U O U
o rq,i qY n g) oY rj n r19/lnu til}lnt lH mobile phase t1r0!1!l ll'ilURUU lii l"d rubing
o tiot14lJ11il1JU 191ril il]y du
2.12 n1YU1{ fl .1 fi 'lv fl ',l :
ilt (pump dampener) r?o
d'rn: orrfiul'lu:vuu
n'r'rud'ud{"f r: plqq
9t:?0cfoufi-l::-?6il rrjdeJu oumn damoener u$o gi'rn:o.t
rdu': t?ix
?^rior2.8 X1A1[Uff?U111111#
solvent ruarLtr?rriu 6 r6o
qiln:niruarudr:1u:vuu
fiilryrrlunr:dr{'ru
p'lush dr s r r d: N ?o q rJ n : aipr a ru a r : #u tYr ti'r nr s rnru fl iy u rtail tA o I
0u tfi[1Jduu t?flJ
d2.14}JO[FIO:11O{ pump
u-rlel
fla9l0s]n
2.15 circuit board "frqn AqflOS1.1
o,Lai3. ilnl::-)ryilil pump head
!93.1 pump plunger seal dfl
fl:ou
njfiau seat'lrri
4t3.2 plunger dnfl5OU .lioil 1.riotil6 uu plunger'lu ri
3.3 pump head 140llJ ilryudngf ido{c.ro{ pump head t#rnju loruovdorur;uq y I r u a I gl - d trty 9)tl"l[tUU[yl'lrlflU ilqyilUtlO? seal Ovqnt191 tq [[nyOyAO{
l4rrat4tlil?luuttuu0urflu tu
- 381s7 -
n1:rrtl'h
3.4 inlet ?1?o outlet check
valve 14d?u
r 4u hlrnju rrsi: y itodrl#rnju o urfr u'lr-J
st : ? 0 do 1J titting I r 0 v femrle'j r r a r I r io r nj u rn^u'hJ n ?o
^o-^__- 4 t4 r ri q r9,/ os'l: Ft1..t : 0 tlJ rljCIuu tu ru nttitl0r<'i'a.i
4. ilnl::?ryilil draw-off
valve
4.1 draw-off valve 1'10?tJ
14:OSt:91
14tlxJ draw-off valve l#rnju
ttiduu seal lJOi drar,v-off valu. lrri4.2 solvent inlet line "fr?fl nJfiuu inlet line 'lrri
s. dq:rnr: lHaHiod'srrv&ar^
n rr ! udt: Rvn tuf"r9l ilfl gl
5.3 Mobile pnase 1utrnfi
'hirfio.rno
5.1 nl: degas ltiO'sgrarge
mobile phase'hitrrurvat
rit mobile phasc 'lil degas r?o sparge d'ru helium rtfrr
ri r'hJ equilibrate T s u u lH *; * fi rluu r y il o drl#n i : d r
helium sparge ri r l#dsr : r a?u ro { solvent ni fi u u hJln rln 9,,
5.2 Iu [9] prime lJtJ prime flru1uri
!fl}J mobile phase
. e4 q '4prime ilru1r ru riori r l'nylo I o tnr g
et : ? 0 do u'iI hifi v'l o s oInrfl 'lu solvent inlet Iine
dt mobile phase lil degas r?o sparge d'lu helium
5.5 inlet ljio outlet check
valve dfliltnu$olixru
t^lailfl9r
11 I fl ?l lJ dV O-l9l check valverdrjilv
9l : ? 0 dO U -l'l
lJ fl I : n : O.: solvent O U t.t [1.1 ]J't y dtL tfr O tl O { n'u
-in]:qqAUn check valve
ololn^Frflt:slnflynourlo.t buffer d check uulu. ldrdo nrn-id, , ,
fl 't : a v n tufr ril u atu plau u old r d'u orsanic solvent luvvo*
d'qr : r ar u s ox r*u'hJ vi.r fi n r : sr n Er y n o u 0 u fi il -r n rfi u s 1 e
v4 rv a 'iluoqjn'lvrTsro.l solvent rrnv buffer fi1#
i'q).6 trlter il09 tU1l?91
solvennriovioal
dt:CIyntuo9lfl'uq
9r : ? 0 do u n I : qrr ri'u r o.r vi o aI ar: ns or Urirs 1rj q rrr o dl
luauu tfilJfit0trlju
lirntrruavotFl filrer fioqjlurroruit0 mobile phase Inodr
filter'lil sonicate lu o N Nitric acid srrild'ruu't : n9.r rrovgvrAi'o
el'tlJei?u methanol oropiolnjduu frit frodlu filter lrris.z rrdrlurirufidrld'
solvent zuarurcirfi'ud rfotdd0iln:ilrududt: [u:vuu
<q4 q orJ u[u].t I tu nt: ?t1,1'lu
nlush fir s l dr n io q rJ n : nfru aru a r : riu tir rir n w r fl q1 u r
o{ hinJfioulrri
- 39t57 -
a4
1lful.il dt[1.rE nr:rrd'lql
5.8 pump plungcr seal dnrd.l.ry
fl :ou t3[11ufl l5: ?ry]J1'liqvlfl pump head
njduu seat'lrri
4r5.9 plunger dnfl:OU vorLuiotrJfi uu plunger'lu ri
5.10 solvent lu pu*p
neaa'hi ar rL'r i r| tt c{}J tlilu ltl 9./flu tgl
e{o z.s
s.t t fiornrg'Iu pu-p
head
stils z.tt
5.12 5v1lu'lv,hlr1o.1
prr''p "Erqo
9t9t9t051.1
u tn ttn r^6. solvcnt P'ldlJnU tO llJ9l
rnr o'iqurq 1'{ tv od i r8.:1u
fl15 run gradicnt
6.1 solvent 'lu pump head
'hi arr r: n prarLt{rriu 1d'
q{o z.a
d n ' do.lt6.2 ?1A1 tU d ?U 11 11 I tl.'l
Yu44solvent zud]J[t]nilq U5O
tddloljn:[uzudufl-]5 tuTvuu4q q o
ulJfullt tun'l:11'l I lu
ptusn dt r r t dr N io o rJ n : nfzu d il d I 5 liu tir f,s r'r I il u fl iu ?i rqU
tq y ri c1 |
0u tl.ttltnuu t14xJ
oalA6.3 pump ill.lliltlqunfl 9r:? odou n'1? ?11.11il uon pump
6.4 n1:p.rdrJ1J0{
d-r: nv n'ru14 d'{oonornll ra
pump $Jfl
.rY.i u^[14 [?\]Jn1: p.tdlJU 0.: d]: av atu9{u
- :vil'jl{ pump outlet Irou injector 1#siodru tuU"
yt"o9t1u]elrflur^I]fluunct{ntulu o.o+o fi-l urr 6-12fit 6snr:
utat4roclt^l4lmltryUU0v tilnl tfifll: run gradient zuA tU0ln[qil
Y qY , U I Yo4 qY^ 1 d- fl-1flI: ts tube tlJ dIXJl:CI[[nil€U14'l t1'1[Y{ilq1Jn:iutlfltJrY
dd cl rd s a l49l IyluUulo [14futu [nuflg t1:Y14?1{ pump outlel tlavrbd,t-laa!
injector o d x "l : fi pr r rL n r : 1 dq rJ n : ai ru ail fr il I u r q td n fr qel
' i dr rn rr'ly zi il u A ..r11t11ovrilu ttj t9r tltoiloinufl'l:[?\:J delay volume ttdu
fl'lt run gradient fifin;rJhl
7. n ?ril d'u q.: zu^n il nfi ln ufi
d1[?l9l]J101il pump
r.t d'rdsr:rnr:'luoqt
rf')^u1il
pi'.ro'gr: tnt:'lu alu ri
7.2 pressure transducer'1il,o . ' iar I
0 g [uerl[r11 u {vlrljuflu u
fi:0Y1qa
q{o z.s
- 40t5'7 -
1J[U111 ffrr14E nt:$ti'hd4a4d
8. lJ[dU{91{[0Ufit ?1 pump 8.1 pump seal tr#.t ar : ri r u fu dr { plunger t iir fi I fi o qi 1 u u ?fl }4 u e 1 #rer^t 1 r ri
Prime 5&UUdrs pt.rng"t 1tlrfi;
virlr{ ptung". rTJun lpruiluer sotvent d,ru',rauusluriota i ts|
[An'l nOqqlUUUl,O{ pump head
48.2 pump plunger seal UFI
nanuttuu
njduu s"at'lrri
9,/448,3 indicator rod (ftlu) U9l
nqnuttuu
nJduu indicator rod"lm ri
8.4 1d plunger seal zuPt
lJUt9l
ld ct tcl 9,, 9)
IUOUU seal [14]J t14fif,iFl0'ta
en:t{fi S rlqntnt:1#sru manual injector
rlcunr fltlfi91 nt:trfftna,i,<.i
L XJflI:5?ryUil injector port 1.1 injector port "f rqfl rjfur?onjfi uu seal'lm ri
1.2 syringe needle g"ug" fi
zuurelNqjr6orfinn'jr
injector port seal
ldturotosr{rufinlr{qnd'o r nr:ld needle gu.rg" fifi
r u t q'l r fli in^u hi o y vi t 1 # i n; ector port sea I "f t :" et
n.ffiuu seal'lrri
1.3 syringe Slqn ISU {o
riorJnrorfi)Jrrnnrflu:d
[fluu[nfl1
nJfiuu syringe ttny seal 'lrri
<J,ei1.4lJflr::?ryil?r disk
valve
14tlu valve b{rnju rrnrilfrl#uJfiuu disk valve'luri
< J,a i2. tJfll::?rylJ?l stator 2.1 dngll rotor stator
fia?lJ
cl9) | 4 s .19/ A qv | | vilUUdn!. [l'l[[UUUU tqU try fcclcr gauge [TlO t?l[IUUtillnUqq
q | 6l I I a ul l - L a qs) tA: v?.10u-t [14 [[u u lfl u t1i ?11 fl u.1]'{ilnl: : ?ry}J [14 [1J0ul] rotor
qtstator [14]J
4V2.2 t'l?l{UlUOi rotor stator
dnfl:ou
njduu stator lrri
du4a3. ilflr:T?q[ilm
compresslon screw
3.1 fitting Ha?ilHiotrilu
tn^u'1il
el : ? 0 dou fi tting un y fem.rle nJ 6 uu 1 n rj dr"f rlor
- 41157 -
rleymr cnrfiel nr:ruT'h
q4vd4. ilq?llrnu?nuflr:49'l
I d u I t1r)9l?ou'1.1 IUU qqn?ou'1.1 [91
4 llU]fl 1'lt05Ul1{ peak
zuerufl9l
44.1 injector port 1'150
v^ldU111.11JO.: vent tube QFI
9ru
9t: ?01.t tfl.109lglu
backflush injector
cfOXJ injector
4.2 syringe "flqn rtu loAlddt
14:OilAtU[1ru[[nn[UU;d
[fluu[nndl
trJfiuu syringe rtnv seal'lHil
dv4d4.3 lJatr:?ryil?1 disk
valve
fi\lJ vaive l#tnju ilrnti.:fr1#nJfiuu disk valve 'lxrj
4.4 rotor seal "f r:"et n-lduu seai'luri
4.5 syringe QftFi'u lirnrrtavolFr syringej
9l :'l 0 ff 0 1J ? 1 lJ fl 1 : fl : O.1 d I : A v n I U solvent t tn J n ? 0 U'.l.: 11
[1'tlJ']vd]J
I o I Y u Vtg)Fl : ? 0 dOU ?l solvent [ [n v fl ? O Ul.1 dltJ'l: 0 zu d]Jtl I nU t9l
5. lfl9] carryover s.t drl samplc loop'hi
[Tlu{110
drt sample loop dru mobile phase mnnS{na9l
5.2 syringe t'tio injector
port dnil:n
'l4 ryri,rg" daru',et
drl injector port
d-li5.3 qQdnu potl tlJ0{O1fl
al i gnment P.l 9l il -r'l'l 9l
Align iliofiori'r injector'lu ri
.l { crt)9ll:l{lYl 9 lJCUfllnl:[U{lu autoinjector
rlarnr cl'ilHq ntflrffh
l. autoinjecto.'hjrit'ttu
Jq ufi d'qailr[0u front panel
hidr.rruniuflu
tn 'tntd t- h I
1.1 txJ t9lrfluuilnfl [TI et 5 ? 0 do ufl 't: rfi uu ar u'lrnl
h ra tn rl ^i1.2 tutJfl :Ylld t1"1V,|111
dY 4 tu 14 l
[Ft1[flUUilAn [?\
a i ? 0 d o 1l e{r rH u u rl d'n 1 vl 1 o u ri r q il n : a:'t v'ht r fi t r il 1 o'j r
1 d.: r u 1 d':L r rfi u u fi u{r rfr u u il d'n ly{ d tir q rJ n : ni'tr,'l fi r hi
vi r r r u r r a el vj r re{r r fi I u il n'n 1 v,l fi il iy u r 1 #nJ fi u u u{r rfi u rrld'nh,'lhrri
1.3 fuse 119 nlfruu fuse luri
- 42157 -
rlsurir cTtlfi91 nr:uiT1zu
tn ro2. autoinjector [lJ?11illJ
s i-lFlu111''l9r Ru[[0U liont panel
lirrru
2.1 autoinjector hi
t6orL gioii'l controller
AIfl : ? 0 dO U fl I : [50 ]J 9l O : y ?l ? I { autoiniector flU controller
, | il q9trrovfl'r:a inrl[]1u{uoi IEEE-488 (01 t.ir IEEE-488)
retdtu160ileiovv
oon 0tn#urtiqA?md autoinjector 6nnfs d1 autoinjector
149,/s vt t, ctYd'lUlTCllll{lU tPltPlU t}Jgl0i IV controller lldAi?1
.. 4s4 .v A I q i r ?control ler }J U ryq I (il llJ CI 0 I fl I U ITO tJ 9l O [U U iU y yl qil n 5 iU
v
ri'raolrflea?mriogl 'o
z.z flcyrrorfln'nud'u
oln'tfl
er :'t 0 d o u'j r fi vi o n r r I d'u o I n I fl er^ q'l {d autoi n1 cc tor
d t4'?t:o txl
vlglr ?0dount: a{n tn?1ruqu o'ln'rfl?'il14lJ1y dufluu?nfl15ct y 4 14 ' 9J ll ' I q y tu yqIsllul.l:0 t]J ot llJflnaoi t14]J5ulrfl l14u
I
dAA2.3 3Jfl ?]ilru9r1iR-l9r11
- t4 l9.l a1'1CI i':tu t1^11''l lu0 llfl: 0 I
a^oq'lreset !fl:O.l autoinjector [latt:iln1:lllnlu [hlJ
I
II
I
z.+ rJ : v sr ryo s ldei'r o dr.:
u9l ttJfluvl
Af ^t | | q lu ' q I y u ^ a4 q 9)[u 9] [ [n y u 9t il : v Fl T0 i I crfl ? o u'l r tH u fi
.l u I [nn | [u lJ 1 t 1.,1SU
flqaoryl{
2.5 :vuu144uflra16i
d'rodx'hir,ir.nu
9t9r9r0T'ln
2.6 circuit board "f tqet 9t9r9ros'1.1
ao!<o3. lJnt:5?rylJ tu:uuu 3.1 fitting Hn?ilil?o[ldu
lnu tu
Ft:?odou fitting ltoe femrlc nJfiuulrriti'rdrf pr
3.2 valve seal "frqot rilduu seat
3.3 seal pack "f t:"or trJfiuu seal pack
3.4 fluid pack "irqn nJ6uu fluid pack
d4dq3.5 rlruanlJUqH'r
4o@A-qddpurge autoinlec,tor lfl 0fl I 0A d{qqnU [U [1ilq91
yu^q4dq9r't.{da0lu{rnqil6u11 ton t}1[ilnuu[1ilqn[[nv seal pack
UA3.6 syringe qqnuH:0
"8r:Fl
trJfruu syringe
. a_ oi 4 9)4. lJnt: :?ryu0'lfl :u uu6].1
tllu
4.1 fitting fln?ilH?ottdu
tflu tlJ
9t : ? t do U fitting t tO y fermle nJ 6 u u 1 H ri tir "f r ; el
4.2 fluid valve "ftqrt njduu fluid valve
4.3 needlewash pump
"f r: pr
trJ o' Uu needlewash pump
- 43t57 -
rlonr cTil149l nr:u#lru
4.4 solvent 0'lflU?g
needlewash 'lunn'rq
autoinjector
I
p1.: I r q needleu'ash ldo qjri r n'jr autoinj ector
s. flcyurnr:finpi'rodrslnrda
e]?our{ tilQnaa H:0
gr"l:r.r peat fiorrJnrr
Isu 5.1 injection valve "ftlot purge autoinjection
tliti.r rfr or il cgr r1#qio I r io ril d uu'ulu.ddua
5.2 rljilqflqfl nu tu0.101fl
dd6to}J01.{Rrn[anl tu n?0u].1
A.-A-qdepurgc autoinjeclor [V'l 0fl 'l 09l d{ qnnU tU ['UXJ A 9l
Fr i ? 0 d o il'i I fi n r : n : o I 6'r o d ^i I t rn v solvent o d r'l tl'l il r v a rl
gtu^qdclyn 1u {rfl et uruil ron r?1 rilfi uurfi ri fr n
s.: fivlo.rorflrfllu purge autoinjection
syringe t'tio sample looP ,i'.r fi ,,^l u u u'.r n r srdr ru r 1 u ri 6 n 1 dsr : r o a o u'i r t d o r fi rL r t vr'r
r^it u r.ptum o v r1o l'hj tn^q cJ n I il qru iU 1 n r g (til rfJ u
qiufgr n rfl t rdn {'jril n septum i iuju tn^u'hJ)
dqtdlltdl^ln 0 { D 9r 9r ? 0 u I I 0 I fl lJ ? 9l 9l ? O U 1 { 11 t 3J U t'l I U 9l
tn rd O
5.4 [rJXJlXglFl ? 0U].t lU
oldud9t'rrr14u.1ilc{{qe
J,o ' i rl11
Fr 5 ? 0 d o u fl r : fl { a q n tl 9i I r ru ri I fi r r.r zu rq d'? o u t I t tri "t I
4 r6ly 9)
[1.tlJ t1lflnfl0i
s.s rjirL rsud'todillu vial
'irirfiusvro
el : ?odouil;urgr : ei?odtq'lu vial
s.a ilqtlruilurfiu'Iil dr
1#rfiorqrgcyrnro
ernrur.lrilqh{unuL
s.z d'rodrsrrffqrfiulilAuravdcl-
roo 0'l { 9r ? o u 1.1 il 5 o n q 0 a : I t : ? t u n I : Q 9l 1J 0': synnge
s.s r{ilfi qTrirolu septurn #l fi n d'to d x lil il iir fr{ n l'hi ro r v septum r td q {'j'ladd
autoinjector il6orfi rufi fl fi fl qnI
s.l iliyrrornfl?luei'u
oln'1fl
r d I o a h/j9t : ? 0 do u i'l tL vt o fl ?,l il 9l u o 1 n 1 fl a n I i ?1 autoinj ector
alrlr1.1:o []J
v
et : ? o d o u fl 'r : ri'wi r n r r ru d'u o 1 fl I fl 'i I [ ]1 il 1 v d :J riu s 1 { a 1 T
aY A l4 | 9,/ t4 | I 1Y lu Yq--^l[ry{tul.tTo tu fl1 tilQnflo{ t1'l1J:uttfl[14]J
5.10 nl: align :vlldrril
Fl?ou-1.: zuoila19l
alig'n'lilrj
5.1 1 injector seal "irqol nJfiuu injector seal
- 44t57 -
6. 1ilfi solvent lnor.iru
injector
6.5 autoinjector 14 ufl TY n n
lJfll; purge
fl q r r dtfl el rn ? o r 1 u ri u r^ o'l # r, u l'e n fr r q oi r r tri r-l r r 6 rri rn ri.:n s fi il iuil ri#,to u rionJfi ou rrdr
Aovauq64purge autoinlector [y,l Ofl 1 0 q d{ qq n U tU [1J ]J Q 9l
rdutt9l : ? 0 d 0 1J ? 1 }J n 1 5 n : O { 9l ? O U I n I tn v solvent 0 U 1 n [14 ]J
-l U dlJ
Y u ^ q4 d O V rj d a
0'tu{tnq!ilH I 0n tH [unuulu]JilFl
n1:uiT1r
m In 9y' | | |
6.1 tlJ t9lFlOilO:VH?li
autoinjcctor riu pu-p
lavio:vu'jri injector fil pu-p 'lt{ solvent lmne.iruki
6.2 injection valve OQhFI
9n[[1.1U{
Al y ^t A q rA q y , a lo r a
U q t[A ?lilqln : O i t?j ]J [Tl O [?1 vah'e n R]J fl gl lt[14 U i [9]:iI
v 4q nyt 4 td d1'l I fl U i ft i lJ lJ 6u 1.1 1 t ?1 ry 0 3J l.t 5 0 rlj R U U'l I n ?
6.3 injcction valve backflush autoinjector
m r ntirn rfi ilcyu r1#niorLmionJd uu r rri'ra o!* q
6.4 )Jflt::?.uilntu [u4r4
autoinjector 14 5 0fl 0U 0.1
autoinjector
9r:?tl.i"r:out?j nu ' &
14}J1J I rttrng 11?14'llJ t1'l tlUUtiU
rrnf,lfrod 1#sr:roaou'it fittiue uov femrle ntuluuv
o 4 t4r t, t4 q 'auloinlcctor ryll"A H t O IU 0l 0 0V9lO i [1J R UU tl'l lJ
7. n?r!d'u'lu:vuuq{
n:6u?1}J dtrl.t 9rlJ't otnq
autoinjector
4a-i7.1 t,ljilqqoqnu [u0.10'lnq
d<qlJounln rancl tu 9r ?0u1.1
7.2 injection valve QFlglll purge automJector
u dq qyt a ri Jilrnuifl {il!iulit turyoilH5 0lilnuu?ra?
7.3 tubing 11910 : Y14 ?1.1
dautoinjector nUn0nUU qA
9ru
144vlilnUu tubing ilqqnu
7.4 restrictor loop QOflU
(ov ti'rrnq'jrn? rild'u uil u
d u r da4a q fl ? o u'r.l rJ uiu 1.1'r )
7.4. I itoer loop oon ornriu land'lr{r hJlH rileul#nductrpiffr:vU'jtl autoinjector inlet [[dU autoinjector outlet 6log
vir'lr? solvent 'lrnorn pump rrhq injector outlet tubing
l4 ta dItAU [Hn00nil1i injector inlct tubing 0{Q1JfltflOl waste
t .q.z fladum r rnr: ln n riou r #rd-.^t . A s) - e-:-i7.4.3 di pump ?11i1}J[1l0Ol{ reslrictor loop llCIUfl109di1i
qord'uoonhJ
z.+.+ dr hiar ru r: CI t rr1il iu r r ld l#nj 6 uil restrictor looo
z.s pi'redr.r'hi arrur: cr
ruarutrliiu mobile phase
el : ? o do u n ?1 il dt u 1 : 0'l u n 1 : n s n 1 u Jq U 1 ad'r o d x r rn y
mobile phase o s'lu m a o n ?1 q a o { r rdtti's rn srjr 6'r o d r.l
dtrJ't : o av aru'lu mobile phase ltio'hj tir drrfl u l#rfr o o rsu | 4 t4
9l?0U1.:14 : O[UOUU mobile ohase
- 45/51 -
rTqnr cnrli9l nr:uffl'u
7.6 autoinjector filter Qg
9tU
lircrrrruavornv$o fitt"t
8. [flfl carryover s.t il?ilrn:fifiprrLrn
rnu [u
ta ia a qc., q tjnoil5il]n:vlqe x5o tT sample loop ulJ19lt?lquu
s.z ilcyu rnr: fi fl d'?odr'1 fl : ? 0 dou lfl ufi fl solvent u fr.rfi qr d'ro dr s dlil u caryover
or r rn^erfl iyu I o I n t v u u dl { r{ xJ
8.3 : vuldr.rrfi rLfi iliYr r
niu hirfiqrnlT prime
uiohifi solvent luurq
needlewash rio f.it
anrJ:n li?o needleu'ash
prr*p "ir1n r{lud'u
tirhifi solvent'luzurpr l#rfitl solvent ns'lutrel
needlewash
t1r'hirfiqnr: p.im" 'lv1 prime l&uu needlewash'lril
il | q I t4flr rnt anil:n [vtilnou frit
d'l needlervash pump dr:"or 1#finriovrs
q. finei'redr{zuet vial q. t d'{fi ev ilrutazud'?ot.il'1
ttr IttJf|fl9r0{
Laqo'j9t : ? 0 d o u n 1 : di q q f,l u gl t t [H u n n ? l'1 lJ ?q n ? 0 ul.1 [ tfl t 1
o 'o9r' 9)
l1.1XJ tl'tf|fl9r0i
or^il9.2 ulnfl?OUlnn fl illtJp'l9l
^9l,'lro6lsr i 1 0 do u 1J ? q Ft ? 0 u'l i Fl 9l il I u tl'l lJ 11 lfi 0 I [u L
ol9.3 n'15 align TUU1l111fl{
vla9t?OU'l{ 29ll','ln19l
align'lrri
10.raidutg.)
il5rJl9r:ilqq tilflnno{,i, a ra
I 0. I d.1il91!:tJ lFlS 29l er: ?0c{ouil;rurqr: fid'lfi orYupirrrfi ri':fi?I{u ?erFl?ou1{
rrfiltlmrib?qnd'o.r
10.2 sample looP ttOU
Aqiln I Isyringc Yl ts [ilQfl910i
nJfiuuturprrlo.l sample loop llCIu syringe t#rlrrutvetl
to.: lrinirtutellto'1
syringe hiqnr{o.r
9t5 ?0 doufl l:lrieirt u l gllJ o { syringe iiu sytinge fr 1#
,vt cr tq Y 9)
rrfl tu t14u t14CIfl9ro {q
to.+ rj?lrusirodrslu
uiat'hirfrusilo
fl : ?ocf ouil;u1n: d'?od'll'lu vial
o"|<iI 0.5 lJfl 'l: 5 ?sXJ?1 sYnnge
rdldryOL?J5OtUAUil sYrtnge
to.o pi'rodr{Hfrqrfiu1il4 o I a u d q --ldo 0't { 9l ? o u l.: il to a fl 0 9l 5 I t t'] t u n'l : q q I 0.1 synnge
I 0.7 Innq€UGU'lnlfl'lu vial fiqlqulilfitJrflel
rord'rodrs'lu I ?n fi il rnrfi u hJo o n
nnrur.hl#144'ril
- 4615'7 -
rTiymr cnrfiql nr:ufflr4V
I l. nr:qqHqqTynflv
u0ufi:.1
r r.r 1# viat 'hinndor qY o o 9., a[U vral 9llufl ltlUyU 110 tttt{ngl
r r.z l#r"lrfl n hi nnrflo.:qY I ^IT vral 9l lSJfl 1[[UyUllJO {zut.tOFl
rr.: flne.lrhi6 n ? ? odou'j'Ifl er.hodrsrH lJ'ty fl u
t t.+ rfirufiprr?o:yuunr:
lilnr4rsrufrorJnpr^
9lFl9loTt'1
11.5 fll: align :vtlutitdl
9r?ou'l{ tJawal9l
FlFtq0ryl.l
12. ruilqn{o r z. r ldeJrfl e ryu^e pJrr rfi r nJ fi uu \rl 1deJrfl nryfr q fi rfl u dor rfl q hi'la r.ptu*riddol
[[nyruouurlj:Jaq tHU
n.2}i, viat hiqndorqY o o Y ^[T vral Fl 1]Jfil[[UyUlUOnzuttRgl
riddq[[nylunuu[1J]Jaq tilil
C^trI2.3 septum trfilrfru'hl a : ? o dou'ir hili'dou scotum
hjpiorl#rJrfln
rj is, rIURUU scptum U14O t?JtJ
rid4oIUNUUIUilf,9t IHTJ
12.4 fil1 align :vUuti-16i.t
d'rotirsfiqilnrel
align lrri
nr:r{d to rlryntnr:l#sru cotumn
rJiurr flrfiel ntrrrfr'l,u
1. nrrrLli'ulu :yuud{ luo
n: il ?lrJd'lt14 qlJ't 0tn
fl0nilu
t. t fi nr:qo6'ud "ol.,,nrt
inlet frit H?o column
outlet frit Hionrulu
nonSJu
dtq column inlet frit Hio column outlet frit rionoii'uri
ti'r ei.r fi fl cy r r b{nJfi uu ain rion o d'ru ri
- aAL2 nonlJuldoildnlv{Ao
ruo{0-}nq€urdu
rJ:vfimfinrrr rforfiprr16lud
so'l?1'1 [UnOn]JU
er : ? o dou il : va'u r n rnro I nod'ru riY | ,-_. J q y rj v dfilFtl column efficiency (N) sit tf rilAUUfiOOilU
1.3 tubing qqei'ur-i
tilnUU tubing
- 47t57 -
fleumr trrr1r4 nr:tti"lqt
z. ;rl:x peat fiflcyrt z. t nrod'tri'lil equilibrate equilibrate n o d'rLrfl#tfi swt o riru 1r qj ov 1# t oo rvirqlo.:
rJTru rpr : nr o firu rf u i o ru r n n'i i d1 u n : fi d 1d ion-pairin g
reasent
z,z nodrrianrl:nY v ? u ^a.1
q Y ri u /n 1 { n 0 o il u 14 I n u { il 1J i}, 14'r t 1.'l [lJ O U U fl 0 n ]J U
- aA2.3 flOnlJU[dO]Jdnlv'l et :'r o ci ouil: v fi mfr nrnt o.r n odrLrf
tltnit .olurr.rn efficiency (N) gir [[0u capacity factor (k') 'lri
o rilurnsu.riu oru fu 1#nJfi uu n o drurf
2.41& tubing fiqtute
mio6'n tubing hi6 nio
1d nttmg ttau femile 'hi
tll u'lg fIlJ
vit system bandspread test tirzua'hjoqjlurnruduorLfu 1#
9t:?0dou tubing tlio fitting ltou femrle fild ortovdo.r
ril6uu1nri
at td2.5 qiu?r Qtr il 0.1lljauu
Ail A ^['il column hcater t]l 0 r-l lU rllJ qfu H 8iJyv
vi'sfi nr : ril 6 uu o at N nfi o v ri r'ld retention ti-e nJ fi u u'hJqq
srr:r.rfi tt rlcynrnr:'l#sru detector
qflJfIJfi I cnrfirl nr:ufrlr
1. dctcctor hiilrsru lnufi
vl'onlttng front panel 'hi
lir{rurtruriu
u tA I u1. 1 detector tlJtSOugl0f,iU
t0 r9Jt?\1,'11
a : ? 0 do u'it at u'lrnl rfi u u ril rl d'n 1 vl r r0 u detector 0 u 1.1
5)
flnfl0{n rd tn r9 j
1.2 ttJiln:Vttd t1''111111
Y 4 to t4 |
rnl[duuilnn tTl
a : ? 0 d o u re{r rfi u r rJ #n lv,l I e u d r e il n : nflrhil r fr r tri 1 o'i rq9 t1 , d A tt o ,g , ,; y - t---{vl-.t-e{-14-!t,r{tu tgrutlduul]latlcfuuuon tTlu 0]qilfl:tu tll1\|l t]J
li r s r u r r aq vir rdr rfi u u il d'n lv{ fi fl su m r l#nj fi o u teir rfi u rrJd'n1r41uri
1.3 fuse 1'lFl n]fiuu rus"'htri
ll ro s i2. detector [ilm'l{1U t9lUT
fi'nnruttnv front panel
yl'1.1'1u
o clg)
2.1 detector Yll{]URlU t9l
i^n'r: n ?uE lJ R'l uU O fl lllJ
d tA1)n'l:ROFI keyboard t?
c) d clYt dtit keyboard 1J-o {fl 1: n?U EilQna 0n til il nqo 0n kcyboard
| [n v a : ? t d o il dt detector d'l u'l : 61 li'l { r u dr ud": ro s'l dalnl
1..i:o tu
da42.2 il fl ?-ltJ ru grv'l CI l9lvl
v ln r9.l Ailn{ iru t1^l1 l't1J0{[915 0{
reset rn?ol detector t[avt?il nl: dr.rrulr ri
2.3 circuit board "irqot gl9rFtosl{
3. detector'hiprolau osgio
fl"r:n?ilnruntuuon
n tA I v3. I detector [1J[$01Jfl OnU
controller
9t : ?0 do 1J fl I : 16o rL gi o : v ri 1 s autoini ector ri'u controller
r rn v n r : pi's ri'u ril xj n I o { IEEE-48 8 td'rtd IEEE-48 8 )
3.2 circuit board 'irqo 9t99t0s'.I.1
- 48t57 -
r1qnr cnrfiel nr:u#lrun ra 2i
4. lamp []Jaq il:o- t4 ro
referencc energy []JYll{1U
4.1 fuse Ulgl tujdou tus""lrri
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Andrea Weston, Phyllis R. Brown. 1997 . HPLC and CE Principie and Practicc. W.B. Saunders company USA,
Calilbmia. 280 p.
Guide to successful operation of your LC system. 1994.Waters. 34 Maple Street, Milford, MA 01757, 022318TP,
revision 1.5194.
- 57t57 -
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