Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 1
Abs ac
Due o he inc easing en i onmen al awa eness o socie y and adminis a ion, a g ea
numbe o egula ions became e ec i e o e he las yea s in o de o p ese e na u al
esou ces es ic ing and limi ing indus ial was e, especially when spills a ec aqueous
sys ems. This ac has con ibu ed o he de elopmen o a la ge amoun o esea ch
p og ams o come ac oss new me hods and p ocesses o moni o and educe con aminan s
p esen in was ewa e .
Among he a ie y o con aminan s p esen in indus ial e luen s, hea y me als a e he mos
haza dous as his compounds a e biomagni ied and can each human o ganism. One o he
me hods de eloped o educing hea y me al concen a ion in was ewa e is bioso p ion.
Bioso p ion p ocess moni o ing has led o he de elopmen o senso a ays o elec onic
ongues. These kinds o senso s equi e exhaus i e aining h ough he analysis o huge
se s o s anda ds, which is ime, e o and eagen consump i e.
This p ojec is add essed on he op imiza ion o a Sequen ial Injec ion Analysis (SIA)
p o o ype buil o p epa e au oma ically andom gene a ed known aining s anda ds and
moni o biop ocess abso p ion o model senso ’s esponse.
In his phase o op imiza ion a minia u e spec ome e is assembled o he SIA ubing o
moni o low esponse in eal ime o a colo an solu ion. Spec oscopic analysis also allows
moni o ing aces o eagen emaining on he sys em.
Calib a ion and cleaning ou ines will be designed o ensu e ep oducibili y. Mo eo e ,
au oma ic p epa a ion o s anda ds will be discussed.
p. 2 Repo
Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 3
Table o con en s
ABSTRACT ___________________________________________________ 1
TABLE OF CONTENTS _________________________________________ 3
1. GLOSSARY ______________________________________________ 7
2. PREFACE ________________________________________________ 9
2.1. P ojec backg ound ........................................................................................ 9
2.2. Incen i e ......................................................................................................... 9
3. INTRODUCTION __________________________________________ 10
3.1. Objec i es ..................................................................................................... 10
3.2. P ojec Scope ............................................................................................... 10
4. FLOW ANALYSIS _________________________________________ 11
4.1. Wha is low analysis? .................................................................................. 11
4.2. Analy ical p ocedu e au oma ion .................................................................. 11
4.3. Flow Injec ion Analysis (FIA) ........................................................................ 12
4.4. Sequen ial injec ion analysis (SIA) ............................................................... 13
5. ULTRAVIOLET-VISIBLE SPECTROSCOPY ____________________ 15
5.1. An o e iew on spec oscopy ....................................................................... 15
5.1.1. Elec omagne ic adia ion ................................................................................ 15
5.1.2. Radia ion In e ac ion wi h ma e ..................................................................... 16
5.2. Bee – Lambe – Bougue Law ................................................................... 16
5.2.1. Basic de ini ions .............................................................................................. 16
5.2.2. Bee ’s Law ...................................................................................................... 17
5.2.3. Limi a ions o Bee ’s Law ................................................................................ 18
5.3. Spec oscopic de ec o s ............................................................................... 18
5.4. Sample Cells ................................................................................................ 20
6. SEQUENTIAL INJECTION ANALYSIS (SIA) PROTOTYPE ________ 21
6.1. Mul iBu e e 2S ............................................................................................. 22
6.2. Main Mani old ............................................................................................... 23
6.2.1. Holding Coil ..................................................................................................... 23
6.2.2. Mul i al e ........................................................................................................ 23
6.2.3. 3-way al es ................................................................................................... 24
6.2.4. Mixing Cell ...................................................................................................... 25
6.2.5. Debubble ....................................................................................................... 25
p. 4 Repo
6.3. In e ace: LabVIEW ...................................................................................... 25
6.3.1. Sc ip s and expe imen s................................................................................... 26
6.3.2. P og amming sc ip s ........................................................................................ 26
6.3.3. Execu ing sc ip s and expe imen s................................................................... 28
7. FLAME S SPECTROMETER ________________________________ 30
7.1. Componen s................................................................................................. 30
7.1.1. De ec o ........................................................................................................... 30
7.1.2. Sample cell ...................................................................................................... 31
7.1.3. Ligh sou ce ..................................................................................................... 31
7.1.4. Op ic ib e connec o s ...................................................................................... 32
7.2. OceanView so wa e .................................................................................... 32
7.2.1. S a ing OceanView ......................................................................................... 32
7.2.2. Acquisi ion pa ame e s..................................................................................... 33
7.2.3. Re e ence and da k spec um .......................................................................... 34
7.2.4. Wa eleng h selec ion ............................................................................ 35
7.2.5. S o ing da a ............................................................................................ 36
8. EXPERIMENTAL PROCEDURE _____________________________ 37
8.1. Reagen s ...................................................................................................... 37
8.2. Calib a ion Me hod ....................................................................................... 38
8.2.1. Ex e nal Calib a ion .......................................................................................... 39
8.2.2. In e nal Calib a ion ........................................................................................... 39
8.3. S anda ds p epa a ion ................................................................................. 39
8.3.1. Phenol ed s ock solu ion ................................................................................. 39
8.3.2. Sodium hyd oxide solu ion 0.1 M ..................................................................... 40
8.3.3. Sodium hyd oxide ca ie solu ion .................................................................... 40
8.3.4. S anda d solu ions ........................................................................................... 40
8.4. De ec ion sys ems cha ac e iza ion pa ame e s .......................................... 40
8.4.1. Limi o de ec ion (LOD) ................................................................................... 40
8.4.2. Limi o quan iza ion (LOQ) .............................................................................. 41
8.5. Dispe sion coe icien ................................................................................... 41
9. RESULTS AND DISCUSSION _______________________________ 43
9.1. Phenol ed cha ac e iza ion ......................................................................... 43
9.1.1. P elimina y es s .............................................................................................. 43
9.1.2. Spec ome e compa ison ................................................................................ 45
9.1.3. Sample deg ada ion ......................................................................................... 46
9.1.4. Selec ing he se o s anda ds .......................................................................... 47
Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 5
9.2. Ex e nal calib a ion ....................................................................................... 48
9.2.1. Sui able low a es ........................................................................................... 48
9.2.2. SIA and Mul i al e p iming .............................................................................. 48
9.2.3. De e mina ion o minimum olume .................................................................. 49
9.2.4. Calib a ion cu es ........................................................................................... 51
9.2.5. Limi o De ec ion and Limi o Quan i ica ion .................................................. 53
9.2.6. Peak Cu es ................................................................................................... 54
9.2.7. Hyd aulic hys e esis ........................................................................................ 54
9.2.8. Cleaning he sys em........................................................................................ 55
9.3. In e nal calib a ion ........................................................................................ 55
9.3.1. Mixing cell p elimina y es s ............................................................................ 55
9.3.2. Calib a ing om 20 ppm s anda d ................................................................... 57
9.3.3. Calib a ing om s ock solu ion ........................................................................ 57
9.3.4. Calib a ing h ough successi e dilu ions ......................................................... 58
9.3.5. Cleaning he mixing cell .................................................................................. 59
10. BUDGET ________________________________________________ 61
11. ENVIRONMENTAL CONSIDERATIONS _______________________ 64
CONCLUSIONS ______________________________________________ 65
FUTURE RECOMMENDATIONS _________________________________ 66
ACKNOWLEDGEMENTS _______________________________________ 67
BIBLIOGRAPHY ______________________________________________ 68
Bibliog aphic e e ences ........................................................................................ 68
Addi ional bibliog aphic e e ences ........................................................................ 69
TABLE OF FIGURES __________________________________________ 71
TABLE OF EQUATIONS _______________________________________ 74
TABLE INDEX _______________________________________________ 75

Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 7
1. Glossa y
A g: A e age
C: Closed
DQA: Da a Acquisi ion Sys em
FIA: Flow Injec ion Analysis
LOD: Limi o De ec ion
LOQ: Limi o Quan iza ion
NO: No mally open
NC: No mally closed
O: Open
PR: Phenol Red
S/N: Signal o noise a io
SIA: Sequen ial Injec ion Analysis
UV: Ul a iole
UV-Vis: Ul a iole Visible
Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 9
2. P e ace
2.1. P ojec backg ound
The p esen Deg ee Final P ojec is a con ibu ion o he esea ch p ojec : “Desa ollo de
Tecnología a Escala Pilo o pa a Depu ación de Aguas Con aminadas con Iones Me álicos
median e Residuos Ag oalimen a ios (TECMET)” unded by Minis e io de de Economía y
Compe i i idad, Mad id, 2013-2015. P ojec CTM2012-37215-C02-02 and o he esea ch
p ojec “SINTESIS VERDE DE NANOPARTICULAS METALICAS A PARTIR DE AGUAS
ACIDAS DE MINA Y EXTRACTOS DE RESIDUOS AGROALIMENTARIOS” unded by
Minis e io de Economía y Compe i i idad, Mad id and FEDER unds, EU, 2016-2018. P ojec
CTM2015-68859-C2-2-R (MINECO/FEDER).
2.2. Incen i e
This p ojec ’s incen i e is se ing he ope a ion condi ions and ob aining ep oducible ou ines
o elabo a e au oma ically s anda ds au oma ically by using a s i e cell implemen ed in he
SIA p o o ype.
The e o e, low UV-Visible spec oscopy will be u ilised o moni o colo an eagen s eams,
in o de o op imize he SIA pa ame e s.
Pág. 16 Repo
magni ude. I is common o di ide he elec omagne ic spec um in o di e en egions as
shown in Figu e 5-2
Figu e 5-2 Elec omagne ic spec um (Ha ey 2009)
The impo an egions o UV- isible spec oscopy a e ul a iole and isible spec ums.
5.1.2. Radia ion In e ac ion wi h ma e
Acco ding o (Ha ey 2009), he phenomena o abso p ion is p oduced because o he
a enua ion o a adia ion’s in ensi y a selec ed wa eleng hs when a beam goes h ough a
sample. Thus, some o he pho ons a e abso bed by a sample and hei ene gy is ans e ed
o elec ons, p omo ing hem o a highe ene gy exci ed s a e. Fo molecules, he ene gy
equi ed o elec onic exci a ion lies in he isible and UV anges. Molecules possess se e al
possible o a ional and ib a ional s a es, so he abso p ion is p oduced o e a wide ange o
wa eleng hs, which is called an abso p ion band.
Spec oscopic measu emen is possible only i he pho ons in e ac ion leads o a change in
one o mo e o he cha ac e is ic p ope ies o elec omagne ic adia ion: ene gy, eloci y,
ampli ude, equency among o he s.
5.2. Bee – Lambe – Bougue Law
5.2.1. Basic de ini ions
(Robinson e al. 2005) de ine he adian powe P o a beam o ligh as he ene gy o he
beam pe second pe uni a ea. A ela ed quan i y is he in ensi y I which is he powe pe uni
solid angle. Bo h powe and in ensi y a e ela ed o he squa e o he ampli ude o he ligh
wa e, and he abso p ion laws can be w i en in e ms o ei he powe o in ensi y.

Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 17
When ligh passes h ough an abso bing sample, he in ensi y o he ligh eme ging om he
sample is dec eased. Calling I0 he in ensi y o he beam be o e en e ing he sample, and I
he in ensi y a e passing h ough, he ansmi ance T is de ined as he a io o I o I0.
T ansmi ance is he ac ion o he o iginal ligh ha passes h ough he sample. To s udy he
quan i a i e abso p ion o adia ion is use ul o de ine ano he quan i y, he abso bance A
whe e
Equa ion 5-2
When no ligh is abso bed, I = I0 and A = 0.
5.2.2. Bee ’s Law
Following a simila app oach o (Ha ey 2009), when elec omagne ic adia ion passes
h ough an in ini esimally hin laye o sample o hickness dx, i expe iences a dec ease in i s
in ensi y o dI, as shown in Figu e 5-1
Figu e 5-3 Bee - Lambe law ac o s modi ied om (Ha ey 2009)
The ac ional dec ease in in ensi y is p opo ional o he sample’s hickness and he analy e
concen a ion C. Acco ding o (Robinson e al. 2005), he p opo ional ela ionship be ween
sample hickness ( he pa hleng h) and abso bance a cons an concen a ion was disco e ed
by P. Bougue in 1729 and J. Lambe in 1760.
The e o e, he in ensi y d op can be exp essed as
Equa ion 5-3
Whe e α is a p opo ionali y cons an . In eg a ing he le side o Equa ion 5-1 o e he en i e
sample
Pág. 18 Repo
Con e ing om ln o log, and subs i u ing in Equa ion 5-2 esul s in
Equa ion 5-4
Whe e b is he pa hleng h, usually in cm, and ε is he mola abso p i i y coe icien , which has
uni s o cm-1 M-1. The mola abso p i i y is p opo ional o he p obabili y ha he analy e
abso bs a pho on o a gi en ene gy. As a esul , ε depends on he wa eleng h o he
abso bed pho on.
Equa ion 5-4 es ablishes he linea ela ionship be ween abso bance and concen a ion, and
is mo e commonly known as he Bee -Lambe law, o Bee ’s law.
5.2.3. Limi a ions o Bee ’s Law
Bee ’s law wo ks bes when he concen a ion is less han abou 0.01 M. A high
concen a ions o analy e, i s indi idual pa icles no longe beha e independen ly o each
o he and s a in e ac ing be ween hemsel es. In his case in e ac ions may change
analy e’s abso p i i y.
Ano he impo an ac o is ha abso p i i y depends on he samples’s e ac i e index, which
a ies wi h he analy e’s concen a ion. The e o e in low sys ems, i is signi ican choosing
he igh ca ie solu ion and eagen medium o a oid adding u he e o .
Some ins umen a ion limi a ions also induce de ia ion om Bee ’s law as (Ha ey 2009)
sugges ed. The i s limi a ion is ha Bee ’s law assumes ha he adia ion eaching he
sample is o a single wa eleng h. Howe e , wa eleng h selec o s pass adia ion wi h a small
e ec i e bandwid h.
The second con ibu ion is due o impe ec ions in he wa eleng h selec o ha allows ligh o
en e he ins umen and each he de ec o wi hou passing h ough he sample. This
phenomenon is called S ay Radia ion. Inside his de ini ion i can also be included he
adia ion no being isola ed p ope ly om he de ec o . S ay adia ion minimisa ion will be
discussed la e on.
5.3. Spec oscopic de ec o s
In his sec ion a small o e iew on spec oscopic de ec o s will be explained. Speci ic
in o ma ion abou he equipmen u ilised on his p ojec will be gi en in he nex sec ion.
Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 19
Equipmen minia u isa ion has led o a new e a o modula spec ome e s.(Ha ey 2009)
classi ies hese spec ome e s as Diode A ay Spec ome e s.
Con en ional ins umen s ha e a single de ec o , so only a wa eleng h a a ime can be
moni o ed. The use o many pho odiodes ins ead o a single pho omul iplie esul s in an
a ay o de ec o s ha can eco d an en i e spec um in less han a second.
Figu e 5-4 Flame-S de ec o o e iew (A e 2015)
Figu e 5-4 is an o e iew o he de ec o u ilised in his p ojec . As shown in (A e 2015), ligh
a i ing om he sample e lec s om a mi o as a collima ed beam owa d he g a ing,
whe e i is dispe sed. Radia ion is hen again e lec ed o m a ocusing mi o , which di ec s
adia ion one wa eleng h apa o he de ec o a ay.
One o he ad an ages (Ha ey 2009) epo s o diode a ay spec ome e s is he speed o
da a acquisi ion, which allows o collec se e al spec a o a single sample. Indi idual
spec a a e added and a e aged o ob ain he inal spec um. This signal a e aging imp o es
a spec um’s signal- o-noise a io, smoo hing he da a. Figu e 5-5 shows he e ec s o signal
a e aging.
Figu e 5-5 Le : Non a e aged spec a. Righ : 15 scans a e aged
The signal- o-noise a io (S/N) a e n scans is desc ibed by ¡E o ! No se encuen a el
o igen de la e e encia., whe e Sx/Nx is he signal- o-noise a io o a single scan.
Pág. 20 Repo
Equa ion 5-5
Howe e , he p incipal disad an age (Ha ey 2009) has ound o hese spec ome e s is ha
he e ec i e bandwid h pe diode, on a pho odiode a ay, is nea ly an o de o magni ude
la ge han ha o a high quali y monoch oma o .
5.4. Sample Cells
Samples a e usually in he liquid o solu ion s a e, and a e placed in cells cons uc ed wi h
UV-Visible anspa en ma e ials. A sample cell mus achie e wo majo equi emen s.
I ’s i ial o assume ha cell’s ma e ial mus le all he adia ion o each he sample and
pass h ough i . Despi e his condi ion, no all ma e ials beha e he same way. Fo adia ion
in he isible ange (400-700 nm wa eleng h), ma e ials such as qua z, glass and plas ic a e
app op ia e. When wo king a sho e han 300 nm wa eleng hs qua z o used-silica cells
mus be used. O he ma e ials show a signi ican abso p ion in his ange and in e e e in
measu emen s.
The o he equi emen he cell mus accomplish is chemical compa ibili y. Ma e ial o he cell
mus be chosen o esis he chemical a ack o he u ilised sol en s and samples. (FIAlab
2015) p o ides a able o compa ibili y o SMA-Z-Cells.
When Bee ’s law was de ined in 5.2.2, Equa ion 5-4¡E o ! No se encuen a el o igen
de la e e encia. in oduced he a iable pa hleng h b. Pa hleng h is di ec ly p opo ional o
he analy e’s abso bance, hence inc easing he pa hleng h will yield o highe abso bance,
which is use ul when analyzing dilu ed solu ions o gas samples.
Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 21
6. Sequen ial injec ion analysis (SIA) p o o ype
The p o o ype consis s in h ee pa s: he au obu e e, he main mani old and a compu e . A
schema ic iew o he whole sys em is ep esen ed in Figu e 6-1.
Figu e 6-1 SIA schema ic modi ied om (Núñez 2016)
A compu e is equi ed o command bo h he au obu e e and he elemen s on he main
assembly h ough he u iliza ion o he p og am called LabVIEW (Na ional Ins umen s). Da a
acquisi ion (DQA) om he senso a ay can also be con olled wi h his so wa e. Howe e ,
DQA and senso s will no be discussed in his p ojec .
Al hough he spec ome e is ex e nal o he p o o ype, i has been added o he schema ic. I
is also con olled by he compu e bu uses i s own so wa e de ailed in 7.2.
Figu e 6-2 displays he comple e assembly in he labo a o y.

Pág. 22 Repo
Figu e 6-2 SIA assembly
6.1. Mul iBu e e 2S
C ison Mul ibu e e 2S is he pumping de ice o he p o o ype. I can ope a e wo sy inges
simul aneously, hough only one has been used in his p ojec ( o al olume o 5 ml).
Ope a ion condi ions o he sys em will be explained la e on in sec ion 8.
The u ilisa ion o a mul ibu e e accomplishes one o he mos ele an SIA sys ems
equi emen s. SIA sys ems mus be able o gua an ee bidi ec ional low mo emen . The
bu e e has a 40000 s eps mo o ha mo es a pis on up o down. Tha means he minimum
olume i can mo e is 1/40000 o he sy inge o al olume.
When u ning on he bu e e he mo o ac i a es lowe ing he pis on o he bo om o he
sy inge, illing i , which is why i is ecommended o always ensu e he e’s wa e o a oid
in oducing ai in o he sys em.
Figu e 6-3 Mul iBu e e 2S
Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 23
In igu e Figu e 6-3 he cu en display o he bu e e is shown. On he igh side, he 5 ml
sy inge u ilised o he pa ame e iza ion o he sys em. (Núñez 2016) ound ha lesse
olume sy inges we e mo e accu a e. Each sy inge has wo inpu /ou pu al es. The igh
one is connec ed o a dis illed wa e ank and he le one, o he holding coil. Commonly, he
igh en y is named Ou and he le en y is named In.
6.2. Main Mani old
6.2.1. Holding Coil
I s unc ion is o a oid ha any eagen a i es o mul ibu e e’s sy inges ha could
con amina e he SIA sys em. When a eagen mus be inse ed in o he sys em he bu e e
has o lowe he plunge , aspi a ing liquid om he coil and allowing he en y o eagen s
om he mul i al e po s.
The holding coil is made om 1 mm in e nal diame e PTFE ube coiled o a solid plas ic od
in o de o keep a secu i y and con en ion olume o s o e eagen s and p o ec he pumping
de ice.
(de Lamo 2014) de e mines coil’s olume om he ollowing exp essions:
Equa ion 6-1
Equa ion 6-2
Equa ion 6-3
The cu en holding coil has a o al olume o 6 ml and a ube leng h o 7.64 m. Ano he
holding coil wi h 12 ml and 15 m is also p epa ed in he case o using he 10 ml sy inge.
6.2.2. Mul i al e
The nex componen ollowing he holding coil is he mul i al e. The mul i al e eplaces he
usual o a o al e in SIA sys ems, educing imes swi ching al e channels because o a o
al es only ha e one o a ion di ec ion.
Mul i al e has i e low al es connec ed o a single cen al channel. En ances 1-4 a e
des ined o he inse ion o ca ie solu ion, eagen s and calib a ion s anda ds. Numbe 5 is
connec ed o a 3-way al e whe e he bioso p ion column will be connec ed o, so samples
can be moni o ed punc ually
Pág. 24 Repo
can be moni o ed punc ually.
6.2.3. 3-way al es
The assembly con ains h ee 3-way al es ha allow he low o ake di e en pa hs h ough
he o he componen s. Figu e 6-4 displays he loca ion and nume a ion o he al es, he
holding coil and he mixing cell.
A i s al e, labelled 0, is placed o e he mul i al e and i s cen al po is connec ed o he
po 5. The bioso p ion column is connec ed o ano he po and a was e line on he
emaining. This al e allows in oducing samples in o he sys em o be analized.
Figu e 6-4 SIA on iew
The second al e, 6, is connec ed o he exi o he mul i al e and o he hi d al e. The
emaining po is cu en ly unused hough i was planned as a was e line.
Finally, he hi d al e, 7, connec s o he mixing cell and o he debubble . Ac i a ing he
al e ia so wa e he s eam goes owa ds one o hese componen s, blocking i s way o he
o he . As shown in Figu e 6-5, al es 0, 6 and 7 ha e an open en ance, a no mally closed
en ance and a no mally open one.
Figu e 6-5 Val e posi ions ( on iew)
The cen al po is always open. Upon ac i a ing he al es, he en ances swi ch o he ON
s a us, opening he no mally closed po and closing he no mally open. This way low pa hs
a e de e mined h ough he SIA sys em. NC and NO po s a e placed as in Figu e 6-5 o
Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 25
al es 6 and 7. Fo al e 0, he en ances a e in e ed, so he le one is he NO and he igh
one is he NC.
6.2.4. Mixing Cell
A s i e cell was designed o eplace he eac ion coil. The cell is made o PMMA and has a
olume o 15 ml. Mixing is p oduced ac i a ing he mo o wi h a neodymium iman loca ed on
he bo om compa men , ha induces mo emen o he s i ing ba on he cell. Mo o powe
can be modi ied mo ing he knob nex o he cell.
Figu e 6-6 Mixing Cell
Reagen s and ca ie solu ion mus be pumped om he holding cell o he s i e cell. A e
he pumping, he agi a ion is ac i a ed by he compu e un il he mix u e is comple ed, and
hen mus be aspi a ed back o he holding coil in o de o be sen o he sample low cell
a e wa ds.
6.2.5. Debubble
A memb ane debubble is placed be ween al e 7 and he de ec ion sys em in o de o
elimina e he maximum amoun o bubbles con ained in he low s eam and a oid
in e e ences on signal measu ing.
6.3. In e ace: LabVIEW
A use - iendly in e ace has been designed in LabVIEW o ease he unde s anding o he
commands p og ammed as well as p o iding a isual display in o ming he s a us o he
execu ion. The c ea ed in e ace also allows con olling and eading g aphically he da a
ob ained ia he senso a ay. Howe e , his unc ion will no be discussed in his p ojec .
Pág. 32 Repo
Figu e 7-4 DH-mini UV-VIS-NIR Ligh sou ce
7.1.4. Op ic ib e connec o s
Two op ic ib e QP450-1-XSR connec o s a e u ilized o ansmi ligh om he sou ce o he
cell and om he cell o he de ec o .
7.2. OceanView so wa e
OceanView is a Ja a-based spec oscopy so wa e by Ocean Op ics, and is capable o
con olling any Ocean Op ics USB spec ome e . The p ocedu e o ob ain spec oscopy
abso bance measu emen s will be de ailed in his sec ion.
7.2.1. S a ing OceanView
Be o e execu ing he p og am, make su e he spec ome e is connec ed o he compu e . I
no , he so wa e will simula e a de ice based on he selec ed wo king op ion. Upon
launching he p og am, he spec oscopy applica ion wiza d sc een will igge showing a g id
o nine wo king modes, as can be seen in Figu e 7-5.
Figu e 7-5 Spec oscopy applica ion wiza d

Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 33
Selec ing Abso bance (concen a ion) UV-Vis abso p ion measu emen s can be execu ed.
The o he op ions a e no explo ed o his applica ion. When Abso bance (concen a ion) is
selec ed, Concen a ion Choice sc een pops ou o e ing h ee op ions as shown in
Figu e 7-6 Concen a ion Choice
“Abso bance only” mode only displays and s o es abso bance da a. “Bee -Lambe law”
mode allows calcula ing concen a ion a a ixed wa eleng h once all coe icien s in Bee ’s
law a e known. “Calib a e om solu ions o known concen a ion” allows s o ing abso bance-
concen a ion da a pai s and c ea e a calib a ion cu e. Abso bance only mode is selec ed as
i allows a be e moni o ing o spec al da a o calib a ion han calib a ion mode. Howe e ,
he las was used b ie ly du ing he p elimina y es s.
7.2.2. Acquisi ion pa ame e s
Choosing he wo king me hod will led o he Se Acquisi ion Pa ame e s sc een, shown in
Figu e 7-7. In his sc een he use de ines he pa ame e s u ilised o ob aining spec al da a.
On he igh side o his sc een, he use can obse e he cu en spec oscopic lec u e.
“In eg a ion ime” de ines he amoun o ime u ilised o calcula e a single spec a, i s de aul
alue is 100 ms. Clicking in au oma ic, Ocean View will calcula e in eg a ion ime se ing i o
he 85% o he spec ome e ’s dynamic ange.
“Scans o a e age” se s he amoun o scans aken o a e aging in o de o inc ease he
S/N, de ined in sec ion 5.3. In eg a ion ime and he numbe o scans o al p ocessing ime is
se o sum less han 1.5 seconds, as acquisi ion s a s o delay conside ably o e his
amoun o ime.
Pág. 34 Repo
Figu e 7-7 Se Aquisi ion Pa ame e s
7.2.3. Re e ence and da k spec um
Figu e 7-8 S o e Re e ence and Backg ound spec um
Once acquisi ion pa ame e s a e de e mined, e e ence and da k spec ums mus be s o ed
in o de o be sub ac ed om he sample’s spec um in o de o ob ain only analy e’s
esponse. Sc eens om Figu e 7-8 appea in o de o s o e spec ums. Re e ence spec um
is selec ed p essing he yellow ligh bulb when he p ope lamp has been selec ed. Da k
spec um is cap u ed closing he shu e on he ligh sou ce while he lamp is on.
When he spec ums a e s o ed, a new window appea s on sc een showing he esul an
calcula ed spec um. This spec um is ob ained h ough Equa ion 7-1.
Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 35
Equa ion 7-1
Whe e Aλ is he simple in ensi y a wa eleng h λ, Dλ is he da k o backg ound in ensi y a
wa eleng h λ and Rλ, he e e ence in ensi y a wa eleng h λ.
7.2.4. Wa eleng h selec ion
When wo king in abso bance only mode, o ob ain all measu emen s du ing a pe iod o ime
a a ixed wa eleng h is in e es ing o wo k in s ip cha mode. This allows isola ing he
signal o he desi ed wa eleng h and plo s a g aphic wi h all acquisi ion da a poin s.
Figu e 7-9 Wa eleng h selec ion
Figu e 7-9 shows he selec ion wa eleng h sc een whe e he desi ed wa eleng h is selec ed.
Wa eleng h selec ion will be discussed in 9.1.
Pág. 36 Repo
7.2.5. S o ing da a
Figu e 7-10 Da a sa ing con igu a ion sc een
Da a sa ing wiza d, displayed in Figu e 7-10, pe mi s con igu ing he ile o ma o he sa ed
da a, he a ge di ec o y whe e iles will be alloca ed, he ile name and an au oma ic su ix
gene a o o mul iple iles o he same expe imen . In o de o sa e all spec al da a inside
he same ile, Time Se ies (column da a) o ma will be selec ed. Sa ed da a will appea
abula ed in h ee columns: acquisi ion ime, ac i e pixels and abso bance alue.
Each ow will con ain a single acquisi ion ime and he measu ed abso bance a ha ime.
O he ile o ma s gene a e mul iple iles o each measu emen , which is useless o long
unning expe imen s as a la ge amoun o iles has o be p ocessed.
This sc een also allows con igu ing he equency da a is w i en on a ile, and p og amming
he du a ion o ile w i ing.
Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 37
8. Expe imen al P ocedu e
In his sec ion, he expe imen al p ocedu e u ilised will be de ailed. In o de o be capable o
moni o ing low mo ing h ough he SIA p o o ype’s ubing, a colou ed eagen solu ion will
be analysed by UV-Visible spec oscopy.
8.1. Reagen s
A colo an solu ion o Phenol ed will be employed as he op ic eagen . I s use o dispe sion
analysis in low sys ems has been desc ibed in (del Ma 2004).
Phenol ed is a weak o ganic acid and a e e sible pH-sensi i e dye. As can be seen in
Figu e 8-1, Phenol ed colou changes when i s p edominan s a e is in acidic o m o in
conjuga e base o m.
Figu e 8-1 Phenol ed in: Le : basic medium Righ : acid medium
Fo his p ojec , phenol ed will be used in i s basic o m whe e i s maximum abso p ion is
inside he 550 nm and 560 nm ange. (del Ma 2004) se s measu emen s a 550 nm while
(Sochacka 2015) wo ks a 559 nm. (Sochacka 2015) se s he wa eleng h a e analyzing
phenol ed ull abso bance spec um o bo h basic and acidic o ms in di e en medium as
shown in Figu e 8-2. As a pa o he expe imen al p ocedu e, phenol ed solu ions will be
cha ac e ized o selec he app op ia e wa eleng h.
To p ese e phenol ed in basic o m, al solu ions will con ain sodium hyd oxide 0.1 M as
sol en , and sodium hyd oxide 10-5 M will be he ca ie solu ion.

Pág. 38 Repo
Figu e 8-2 Phenol ed abso p ion spec um (Sochacka 2015)
8.2. Calib a ion Me hod
(Robinson e al. 2005) de ine calib a ion as he p ocess o es ablishing he ela ionship
be ween he measu ed signal and known concen a ions o analy e. A e es ablishing his
ela ionship, he concen a ion o he analy e in an unknown sample can be calcula ed
measu ing i s esponse.
Figu e 8-3 Reagen aspi a ion p ocedu e modi ied om (Núñez 2016)
Figu e 8-3 displays a gene al p ocedu e o in oduce samples on he sys em. Fi s , he
bu e e is loaded wi h dis illed wa e om he dis illed wa e ank and is dispensed h ough
he mul i al e, cleaning he ubing om p e iously u ilised solu ions. Nex , a olume o ca ie
is aspi a ed h ough mul i al e po 4. Then, samples a e aspi a ed ia one o he en ies 1-3
o he mul i al e, as shown in Figu e 8-4 , mo e ca ie solu ion is injec ed a e wa ds.
Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 39
Figu e 8-4 S anda d en ies
The aspi a ed olume is s o ed in he holding coil. Reagen s a e in oduced be ween wo
olumes o ca ie o p e en high deg ees o dispe sion. Finally, i is dispensed o he
de ec ion sys em o he s i ing cell, depending on he kind o de e mina ion.
8.2.1. Ex e nal Calib a ion
S anda ds a e injec ed in o he sys em u ilizing po s 1-3 o he mul i al e, while he ca ie
solu ion is aspi a ed om po 4. Each s anda d is in oduced sepa a ely, ha is, he nex
solu ion is no in oduced in o he sys em un il he las has been sen o he de ec o .
8.2.2. In e nal Calib a ion
A concen a ed s anda d will be injec ed and hen dilu ed o he same concen a ion as he
s anda ds u ilised in 8.2.1. Following a simila p ocedu e o Figu e 8-3 ca ie is aspi a ed
om 4 and samples om 1-3. Reagen s a e dispensed in o he mixing cell and hen he es
o he needed olume o he dilu ion is added om ca ie solu ion, o p ese e basic
medium.
8.3. S anda ds p epa a ion
Calcula ions o each solu ion can be ound in Annex A.
8.3.1. Phenol ed s ock solu ion
Following he p ocedu e desc ibed by (del Ma 2004) and (Vinde oghel 2005), a s ock
solu ion o phenol ed 400 ppm in NaOH 0.1 M was p epa ed.
0.100 g o phenol ed is weighed and added o a olume ic lask o 250 ml. Then 1.00 g o
sodium hyd oxide pelle s is weighed and dissol ed in Milli-Q wa e , and hen added o he
olume ic lask. Make up o he ma k wi h Milli-Q wa e .
Pág. 40 Repo
8.3.2. Sodium hyd oxide solu ion 0.1 M
4 g o sodium hyd oxide a e weighed on a beake and hen dissol ed in Milli-Q wa e . Then i
is added o a olume ic lask o 1000 ml. Then i is made up o he ma k wi h Milli-Q wa e .
8.3.3. Sodium hyd oxide ca ie solu ion
25 μl om he 0.1 M NaOH solu ion a e pu on a 250 ml olume ic lask. Then Milli-Q wa e
is added o he ma k.
8.3.4. S anda d solu ions
S anda d solu ions a e p epa ed aking aliquo s om he Phenol Red s ock solu ion and
sol ing hem in sodium hyd oxide 0.1 M in 100 ml olume ic lasks. This da a is dispalyed in
Table 8-1. Howe e , a di e en se o solu ions was made o cha ac e ize phenol ed
abso p ion spec um. O iginally a se o solu ions we e a ailable om an olde 4000 ppm
s ock solu ion and we e u ilized as es s o s a designing he i s p ocedu es.
S anda d
Concen a ion
(ppm)
Aliquo olume
(ml)
NaOH olume
(ml)
1
0.25
99.75
2
0.5
99.5
4
1
99
8
2
98
12
3
97
16
4
96
20
5
95
Table 8-1 S anda d solu ions
8.4. De ec ion sys ems cha ac e iza ion pa ame e s
8.4.1. Limi o de ec ion (LOD)
De ec ion limi is de ined by he In e na ional Union o Pu e and Applied Chemis y (IUPAC)
in (Nič e al. 2009), as he smalles concen a ion o analy e ha has a signi ican ly la ge
signal han he signal om a sui able blank.
The LOD in mos ins umen al me hods can be ansla ed in o he ollowing exp ession:
Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 41
Equa ion 8-1
In Equa ion 8-1, SDL is he analy e’s de ec ion limi , Sb is he a e age signal o blank, σb he
blank’s s anda d de ia ion and z is an in ege .
8.4.2. Limi o quan iza ion (LOQ)
(MacDougall and C umme 1980) es ablish as a minimum c i e ion ha he egion o
quan iza ion should be clea ly abo e he limi o de ec ion. Equa ion 8-1 also desc ibes he
loca ion o he LOQ.
Figu e 8-5 Regions o de ec ion and quan iza ion (MacDougall and C umme 1980)
Howe e , a dis inc ion on z should be made in o de o obey LOD’s and LOQ’s de ini ions.
Figu e 8-5 shows his di e ence: he bo de o he egion o de ec ion is a a dis ance 3σb
om he blank a e age epo ed signal, and he e o e, all alues ha all below should be
epo ed as non-de ec ed. Values o e 10σb a e conside ed on he egion o quan iza ion.
8.5. Dispe sion coe icien
Dispe sion in low sys ems was explained in sec ion 4.3. In his segmen , dispe sion
quan i ica ion will be add essed.
In bo h FIA and SIA echniques, zone sequencing and mu ual dispe sion o he zones a e he
key ope a ions, as (Gubeli e al. 1991) s a e. Fo eagen -based chemis ies, being op ical
me hods amongs hem, a mix be ween sample and eagen zones mus be done in a
sui able p opo ion and hus a medium dispe sion has o be achie ed. On he o he hand,
conduc i i y measu emen equi es limi ed dispe sion.
The dispe sion coe icien D is de ined in (Gubeli e al. 1991) as he a io o he concen a ion
o he sample ma e ial be o e (C0) and a e (C) he dispe sion p ocess has aken place,
Pág. 48 Repo
I is isible ha abso bance spec um o s anda ds 30 and 40 ppm is e y noisy. Bee ’s law
wo ks bes o dilu ed solu ions whe e a linea co ela ion be ween abso bance and
concen a ion can be es ablished. Commonly, abso bance alues supe io o 1.5-2 a e
conside ed ou side he linea ange, so bo h s anda ds we e disca ded. The calib a ion cu e
p esen ed in Figu e 9-9 shows how linea i y s a s o d op.
Figu e 9-9 O iginal s anda ds’ calib a ion cu e
Howe e i was decided o keep he 20 ppm s anda d and in oduce wo new poin s
co esponding o 1 ppm and 16 ppm in o de o ex end he s udy ange.
9.2. Ex e nal calib a ion
All expe imen s de ailed in his segmen a e pe o med u ilizing he SIA p o o ype. Sc ip s
u ilised o each de e mina ion will be named and he me hodology will be explained.
Howe e sc ip iles will be p esen ed in Annex 1. Spec ome e acquisi ion pa ame e s will
also be de ailed o each de e mina ion.
9.2.1. Sui able low a es
Usually, FIA and SIA sys ems wo k wi h low a es om 1 ml/min o 5 ml/min. (Núñez 2016)
s udied he a ailable and ecommended ange o eloci ies ha could be se on he
au obu e e, and measu ed and abula ed he mos sui able wo king low a es o bo h 10 ml
and 5 ml sy inges. One o he ecommended low a es he es ablished included in his
in e al was 1.2 ml/min. This alue has been selec ed as he low a e o aspi a ion as well
as dispensa ion in all de e mina ions.
9.2.2. SIA and Mul i al e p iming
Be o e unning any expe imen s on he SIA p o o ype, gene al p iming o all al es and
ubes mus be ca ied on. Sys emP iming. x will load he sy inge wi h dis illed wa e and

Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 49
dispensa ions au oma ically opening all al es ensu ing low a e se all componen s.
Addi ionally, a olume is loaded in o he mixing cell and discha ged a e wa ds.
When p og amming bu e e o de s o load solu ions h ough he mul i al e po s i is
p esupposed ha i s ubing is comple ely illed o solu ion. Mul i al eP iming. x discha ges
he sy inge in o he dis illed wa e ank (as he holding coil p e en s any compound om
eaching he bu e e). Nex , a qua e o he sy inge’s o al olume is aspi a ed om each po
o en ies 1-4. Las , dis illed wa e dispensa ions h ough he mainline discha ge he holding
coil un il abso bance d ops o he baseline.
The p ocess spec um is ep esen ed in Figu e 9-10.
Figu e 9-10 Mul i al e p iming
9.2.3. De e mina ion o minimum olume
This expe imen was designed o de e mine he minimum olume ha mus be injec ed in o
he sys em o ob ain he maximum signal in abso bance a he same ime ensu ing ha
dispe sion coe icien ends o he uni , as de ined in sec ion 8.5.
A se o injec ions o he 16 ppm phenol ed solu ion will be analyzed. Injec ed olume s a s
a 25 μl and is pumped di ec ly o he de ec ion sys em. Se e al samples a e injec ed
inc easing by 25 μl he aspi a ed olume un il a inal olume o 1100 μl. The abso bance o
each injec ion will be measu ed o de e mine he minimum olume needed o achie e
maximum abso bance.
To ensu e peaks do no o e lap and all colo an is ca ied ou o he sampling cell addi ional
3.75 ml o dis illed wa e a e dispensed be ween each injec ion. This has been p og ammed
in MinVolumeTes . x .
Fo he spec ome e pa ame e s, in eg a ion ime was se au oma ically o 236.91 ms and
he amoun o scans o a e age was 6. Rep esen ing abso bance, ob ained as he peak
Pág. 50 Repo
heigh o each injec ion, e sus he injec ed olume he cu e in Figu e 9-11.
Figu e 9-11 Abso bance o di e en injec ed olume
Applying Equa ion 8-3 o he poin s ob ained, and de ining A0 as he maximum abso bance
measu ed in Figu e 9-11, he dispe sion coe icien and he a io o abso bance e olu ion can
be s udied o inc easing olumes. Bo h pa ame e s a e plo ed in Figu e 9-12
Figu e 9-12 Abso bance a io and dispe sion coe icien
In o de o de e mine he minimum olume ha shall be injec ed in he SIA p o o ype o
ob ain accep able esponse bo h, dispe sion coe icien and abso bance a io, mus end o 1.
I is obse ed in Figu e 9-12 ha dispe sion coe icien eaches i s limi signi ican ly as e
han abso p ion a io. Fo olumes la ge han 925 μl bo h cu es a e inside a 5%
disc epancy ega ding he heo e ical limi . In u he de e mina ions, sample olumes will be
se o 1 ml.
Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 51
9.2.4. Calib a ion cu es
Calib a ion cu es a e d awn plo ing he peak heigh o he abso bance o each o he
s anda d solu ions. S anda ds a e sequen ially injec ed and he nex is no in oduced in he
sys em be o e i s p eceden has no been ca ied ou .
All he calib a ion cu es p esen ed in his sec ion a e ex e nal calib a ions. S anda ds a e
in oduced in o he SIA and hen a e sen di ec ly o he de ec o sys em. Po 4 o he
mul i al e has always been ese ed o ca ie solu ion. All calib a ion sequences we e
made om he same sc ip which has been e ol ing h ough he analysis o he spec al
cu es.
A olume o 0.5 ml o ca ie is injec ed p io o he colo an aspi a ion in o de o minimize
dispe sion inside he holding coil. Addi ionally, 50 μl o ca ie a e also aspi a ed a e he
colo an injec ion. Dis illed wa e is injec ed a e wa ds o push he solu ions h ough he SIA
mainline. Calib a ion sequence is ound in Calib a ionP ocedu e. x . An addi ional sc ip ,
Calib a ionP ocedu eDualPeak. x was u ilised o a ew de e mina ions ha will be de ailed
la e on in 9.2.7.
Figu e 9-13 Calib a ion cu es Le : 1-20 ppm Righ : 1-16 ppm
One o he undamen al concep s in calib a ion is es ablishing he linea i y ange so he
esul an cu e can be desc ibed by Bee ’s Law. Two calib a ion cu es a e p esen ed in
Figu e 9-13. I can be obse ed ha eg ession coe icien s imp o e excluding he 20 ppm
s anda d in calib a ion as i s a s o d i ou side linea i y. Thus he linea i y ange will be
de ined be ween 1 ppm and 16 ppm.
Pág. 52 Repo
Figu e 9-14 Calib a ion 2016-6-1
Al hough Bee ’s law cu es in e cep (0,0), eg ession i s displayed in Figu e 9-13 and
Figu e 9-14 p esen a y-axis in e cep de ia ion. This de ia ion on he eg ession models is
due o he elec onic noise p oduced du ing he usual ope a ion o he spec ome e .
Baseline oscilla ion will be de ailed in 9.2.5.
Ga he ing all he calib a ion esul s du ing he p ojec , a gene al abso p i i y coe icien can
be calcula ed. Fo a 95% con idence in e al:
Ne e heless, as s udied in 9.1.3, s anda ds decay o e ime, hus abso p i i y coe icien
can be s udied g ouping calib a ions by he amoun o days passed since hei p epa a ion.
Nume ical da a can be ound in Table 9-1 and he in e als plo ed in Figu e 9-15.
Day
Mean
Con idence In e al
1
0.1313
(0.1145, 0.1481)
2
0.1160
(0.1014, 0.1306)
3
0.1226
(0.1161, 0.1292)
4
0.1069
(0.0955, 0.1182)
5
0.1051
(0.0881, 0.1220)
Table 9-1 Abso p i i y con idence in e als
Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 53
Figu e 9-15 Con idence in e als o abso p i i y o e ime
I can be obse ed in Figu e 9-15 ha he mos signi ican d op in abso bance is p oduced
be ween he i s and second days. U ilizing ANOVA me hods o compa e he means o each
g oup, wi h a alue o α = 0.05, i is ob ained ha popula ion means di e wi h a p- alue o
0.015. When compa ing he o he g oups excluding day 1 g oup, o a p- alue o 0.052
means can be conside ed s a is ically equal.
9.2.5. Limi o De ec ion and Limi o Quan i ica ion
A se o blank measu emen s we e egis e ed be ween 15:53 pm o 15:57 pm, s o ing 157
acquisi ions. In eg a ion ime was se au oma ically o 158.64 ms and 8 scans we e
a e aged. Due o he la ge numbe o da a, i is assumable ha he sample’s a iance (s)
app oaches o he s anda d de ia ion o he blanks (σ). Blank’s mean and σ, LOD and LOQ
a e calcula ed in Table 9-2.
Mean
0.0107
S anda d De ia ion
0.0015
LOD
0.0151
LOQ
0.0255
Table 9-2 Blank cha ac e iza ion
54321
0,15
0,14
0,13
0,12
0,11
0,10
0,09
0,08
Day
Abso p i i y coe icien
In e al Plo o Abso p i i y coe icien
95% CI o he Mean
Indi idual s anda d de ia ions we e used o calcula e he in e als.

Pág. 54 Repo
9.2.6. Peak Cu es
When ep esen ing all da a poin s acqui ed du ing a calib a ion in on o ime, peak cu es
a e d awn. Analyzing peak o ms in hese cu es allows con olling i eagen s a e being
adso bed on he ubing walls, and he posi ion o he samples inside he sys em can be
in e ed by peak o m.
Figu e 9-16 Peak cu es compa ison
In he Figu e 9-16, a compa ison o he peak cu es o a calib a ion u ilizing s anda ds 1-4
ppm is shown. I was obse ed in he le cu e ha baseline was ising o e ime, and when
he nex peak a i ed, a alley was gene a ed and hen abso bance s a ed o inc ease. This
phenomenon was due o he samples no being comple ely expulsed om he sample cell.
One o he ails emained in he op ical pa h. In he cu e on he le , 500 μl dis illed wa e
we e dispensed a e he solu ion (cu e on he igh ), In o de o co ec his p oblem 2050 μl
wa e we e dispensed.
9.2.7. Hyd aulic hys e esis
In his sec ion low e ec s will be s udied. I was obse ed ha when au obu e e inished he
pis on mo emen s, low was s ill a ec ed by p essu e and mo ed back when any al e was
pe mu ing. One solu ion o his phenomenon was o pu wai ing imes a e dispensa ions
and aspi a ions. Coding 2 seconds educed conside ably he phenomena.
On he o he hand, ano he s udy was ealized e e ed o calib a ion mechanics. Calib a ions
peaks we e duplica ed and he s anda ds o de was e e sed (downwa ds) o see i he e
was any in luence o i he less concen a ed solu ions p esen ed aces o he highe
s anda ds.
Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 55
Figu e 9-17 Upwa ds and downwa ds calib a ions
Figu e 9-17 shows he calib a ion cu es o an upwa ds calib a ion (s a ing a 1 ppm) and a
downwa ds calib a ion (s a ing a 16 ppm). No di e ences we e obse ed be ween peak
heigh s ei he be ween same concen a ion, same calib a ion and same concen a ion
e e se calib a ion. I is concluded hen, ha bo h calib a ion di ec ions a e independen ly
eliable.
9.2.8. Cleaning he sys em
De i ed om analyzing peak o ms like in 9.2.6, i was ound ha dispensing dis illed wa e
h ough mainline all eagen s can be ca ied ou o he sys em. The same sc ip o p iming
he sys em can be u ilised as a cleaning ou ine. Howe e , passing a 0.1 M HNO3 solu ion
and hen insing is ecommended once a week.
Acid cleaning is ecommended a e long expe imen s o p e en eagen p ecipi a ion,
especially i he low is s a ic o se e al hou s. O he wise, ubing o al es can be obs uc ed
gene a ing p essu e necks when p opelling low, o acuum bubbles in he sy inge when
aspi ing, which can cause al e mal unc ioning and he sy inge glass o b eak.
9.3. In e nal calib a ion
In e nal calib a ion me hods u ilize he mixing cell o dilu e a concen a ed s anda d o he
ex e nal calib a ion s anda ds in o de o being able o compa e magni udes.
9.3.1. Mixing cell p elimina y es s
A se o expe imen s was designed o check he wo king condi ions o he mixing cell. The
20 ppm s anda d was dilu ed o 50% dissol ing i in NaOH ca ie solu ion o a o al olume
o 4 ml. Du ing he i s g oup o ials, i was obse ed ha when dispensing he same
amoun o aspi a ed luid, ail aces emained in he holding coil. Agi a ion was se o 5
seconds. The me hod can be ound in Dilu ion50%M1M4. x , Dilu ion50%M2M4. x ,
Dilu ion50%M3M4. x .
Pág. 56 Repo
(Escudé 2015) designed he ubing o he sys em. Be ween he mul i al e and he mixing cell
he e a e 435 mm o Te lon ubing 0.8 in e nal diame e , a olume o 874.6 μl olume.
(Núñez 2016) s a ed ha al es could be conside ed s agnan so he olume displacemen
inside can be negligible compa ed o he ubing olume. Howe e , ha is only ue when
al es ha e been p imed. Cleaning he mixing cell equi es emo ing all he luid e ained
inside pa h om al e 7 o he mixing cell. An addi ional olume o 1000 μl was decided o be
dispensed o compensa e hese olumes. Howe e , hal o ha olume is dispensed a e he
phenol ed solu ion and he es a e he ca ie solu ion, allowing ca ie solu ion o push
colo an inside he cell.
Po s 1-3 o he mul i al e we e ed wi h he 20 ppm s anda d and en y 4 was ese ed o
NaOH ca ie solu ion. Ten eplicas o each po we e made s a ing po 1 o po 3 and
ano he en eplicas e e sing he o de . Dilu ion esul s a e ep esen ed in Figu e 9-18 and
nume ical da a in Table 9-3.
Figu e 9-18 Ex e nal calib a ion and 50 % dilu ions Mul i al e po s a e labelled M1, M2, and
M3 Le : Ascending o de Righ : Descending o de
Ascending
Descending
Concen a ion
mean (ppm)
S De
Concen a ion
mean (ppm)
S De
M1
9.62
0.14
8.91
0.10
M2
9.72
0.02
9.00
0.11
M3
9.77
0.13
9.09
0.12
Table 9-3 Concen a ions ob ained o 50% dilu ions
Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 57
Be ween bo h se ies o calib a ions, no di e ences a e ound be ween M1, M2 and M3
se ies, as hei means in each calib a ion a e s a is ically equal. Thus he e is no p e e ence
in injec ing samples h ough a de e mined po . The d op in concen a ion is due o he decay
o he s anda ds wo days a e he ascending calib a ion.
9.3.2. Calib a ing om 20 ppm s anda d
Unde he same wo king condi ions, and modi ying he sc ip s u ilised in sec ion 9.3.1, a se
o sc ip s is p og ammed o dilu e he 20 ppm s anda d o he s anda ds u ilised o ex e nal
calib a ion. These sc ip s a e 20 o1. x , 20 o2. x , 20 o4. x , 20 o8. x , 20 o12. x and
20 o16. x .
Two eplicas o wo in e nal calib a ions we e made as o he i s calib a ion ca ie solu ion
addi ion be o e colo an aspi a ion was no added. The calib a ions a e hen ep esen ed wi h
an ex e nal calib a ion in o de o obse e he de ia ions. Cu es a e p esen ed in Figu e
9-19.
Figu e 9-19 In e nal calib a ions using 20 ppm as s ock solu ion
Fo he second calib a ion, he eplicas we e a e aged and compa ed wi h a new calib a ion
made he nex day, as he expe imen las ed all nigh . The e o e, calib a ion 1 app oxima es
mo e accu a ely o 2016-6-7 calib a ion. I can be obse ed also in Figu e 9-19 ha small
de ia ions appea o he leas and mos concen a ed dilu ions. Fo small olumes, colo an
solu ion is mo e a ec ed by dispe sion as abso bance alues all below he ex e nal
calib a ion while o concen a ed samples, abso bance alues a e abo e he cu e.
9.3.3. Calib a ing om s ock solu ion
Simila ly o segmen 9.3.2, he same p ocedu e is applied u ilizing he 400 ppm s ock di ec ly
ins ead o make he dilu ions. Sc ip s u ilised ollow he same ins uc ions changing he
olumes o main ain he dilu ion ac o . These sc ip s a e 400 o1. x , 400 o2. x , 400 o4. x ,
400 o8. x , 400 o12. x and 400 o16. x .
Pág. 64 Repo
11. En i onmen al conside a ions
In his p ojec se e al en i onmen al conside a ions ha e been aken accoun as he
School’s di ec i es indica e o Deg ee Final P ojec s.
Sequen ial injec ion analysis sys ems educe he en i onmen al impac because he use o
eagen s is minimized as he p ocess is au oma ed and con olled elec onically, he e o e,
mo e accu a e.
Elec onic ongues, hough equi ing aining wi h a a ie y o s anda d se s, a e less
agg essi e echniques han a omic abso p ion o o he classical analy ic me hods.
Howe e , du ing he expe imen a ion o he p ojec a la ge olume o esiduals has been
gene a ed due o he deg ada ion o he s anda ds: a se o new solu ions had o be p epa ed
each week. A e ha ing de eloped he p ojec o wen y weeks, an app oxima e olume o
62 li e s o aqueous esidue has been gene a ed. Residual solu ions we e labeled unde he
ca ego y o O ganic Colo an Reagen s.
On he o he hand, he inal applica ion o he p o o ype o moni o ing bioso p ion p ocesses
will a ou he op imiza ion o his echnique in long e m, hus elimina ing and eco e ing
g ea e yields o hea y me als. Bioso p ion p ocesses addi ionally eu ilize ag a ian esidual
o cap u e hese me als.
Consequen ly, he bene i s epo ed in op imizing bioso p ion p ocesses as well as elec onic
ongues ou come he ini ial impac du ing he i s phases o he design and implemen a ion.
The e o e, elimina ing hea y me als h ough bioso p ion educes he isk o biomagni ica ion
and u al esidual a e eu ilised.

Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 65
Conclusions
I was analyzed he in luence o luo escen ligh ning on he measu emen s by co e ing and
unco e ing he sample cell. Spec oscopic da a de e mined ha p ese ing he sample cell
om luo escen ligh ing is equi ed.
Flame S Spec ome e eliabili y was con as ed compa ing calib a ion cu es wi h a
con en ional spec ome e (UV-Mini 1240).
I was de e mined ha expe imen al maximum abso bance wa eleng h was 560 nm and i
was wi hin an accep able 5% e o ma gin om he li e a u e alues.
Phenol ed s anda d solu ion deg ada ion was measu ed h ough he e olu ion o i s
abso bance o e a pe iod o ime. I was obse ed ha concen a ed solu ions decayed
as e .
A minimum olume o sample o 925 μl was de e mined o minimize dispe sion e ec s in o
he ca ie solu ion and he wash solu ion (dis illed wa e ), while limi ing abso bance loss in
less han he 5% o he maximum alue.
1 ppm o 16 ppm linea i y ange was es ablished analyzing he noise le els in spec al da a
and plo ing calib a ion cu es.
Rep oducibili y o ex e nal calib a ions has been p o ed al e ing he o de samples a e
injec ed.
The addi ional olume o compensa e pa h leng h be ween mul i al e and mixing cell was
se in 1000 μl. Mo eo e , ep oducibili y has been es ed, o di e en con igu a ions.
The e o e, I was ound ha injec ing solu ions was independen o he mul i al e po he
sample is loca ed.
In e nal calib a ion dilu ing a 20 ppm s anda d was se and i s eliabili y has been con i med
when compa ing da a o he ex e nal calib a ion.
Sc ip s o s anda d ope a ions including p iming he sys em, calib a ing and cleaning we e
p og ammed and e i ied ob aining peak cu es usual in low sys ems.
Howe e , p og amming in e nal calib a ions using he 400 ppm was no accomplished.
Pág. 66 Repo
Fu u e ecommenda ions
Implemen ing LabVIEW and Ocean View unde he same so wa e in e ace would allow
pe o ming spec oscopic and elec ochemical analysis wi h he senso a ay simul aneously
on he SIA sys em.
Val e 6 is connec ed in a sub op imal way: he no mally open po has a lid o p e en ing
luid escaping. Fo long wo k sessions some luid leakage is p oduced. Addi ionally,
connec ing he mul i al e o he no mally closed equi es ac i a ing he al e o all
ope a ions which inc eases ope a ion imes.
In e nal calib a ion dilu ing concen a ed solu ions should be s udied u he on. Pe haps
es ing on inc easing concen a ions om 20 ppm o 400 ppm can de e mine he maximum
concen a ion in e nal calib a ion wo ks up o.
S i e op imiza ion could be s udied mo e in de ail i he knob ha con ols he mo o had a
e e ence sys em o know he pm.
Addi ionally, a g adua ed scale o olume in he mixing cell would help e i ying
dispensa ions and aspi a ions in he cell.
Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 67
Acknowledgemen s
Fi s o all, I would like o exp ess my g a i ude owa ds An onio Flo ido o p o iding me he
oppo uni y o handling ano he p ojec unde his guidance, and owa ds his cons an
supe ision as well as o p o iding necessa y in o ma ion ega ding he p ojec and all he
pa ience shown especially on he i s weeks p epa ing Phenol Red s anda ds and allowing
me o u ilize addi ional equipmen om academic labo a o ies.
I would also like o show g a i ude o Sa a, my pa en s and my b o he s o suppo ing me
du ing hese yea s s udying Deg ee in Chemical Enginee ing and encou aging me o
con inue ad ancing.
Las , bu no leas , hanks o E ic, Ad ián, José Luis, Anabel, Eli and C is ina o sha ing
expe iences in he labo a o y.
p. 68 Repo
Bibliog aphy
Bibliog aphic e e ences
AVE, D., 2015. Flame Minia u e Spec ome e Use Manual
BAXTER, P.J. and G.D. CHRISTIAN, 1996. Sequen ial Injec ion Analysis: A Ve sa ile
Technique o Biop ocess Moni o ing. Accoun s o Chemical Resea ch [online], 29(11),
515–521 A ailable om: h p://pubs.acs.o g/doi/abs/10.1021/a 950214z
CHRISTIAN, G.D., 2003. Flow analysis and i s ole and impo ance in he analy ical
sciences. Analy ica Chimica Ac a, 499(1-2), 5–8
ESCUDÉ, B., 2015. (SIA), Op imi zació d’un sis ema de moni o i zació de p ocessos basa
en anàlisi pe injecció seqüencial
FIALAB, 2015. Fibe Op ic SMA Z-Flow Cell Manual Design
GUBELI, T., G.D. CHRISTIAN and J. RUZICKA, 1991. Fundamen als o Sinusoidal Flow
Sequen ial Injec ion Spec opho ome y. Analy ical Chemis y, 63, 2407–2413
HARVEY, D., 2009. Analy ical Chemis y 2.0, 810
KIKAS, T., 2014. In oduc ion o Flow Injec ion Analysis ( FIA ) De e mina ion o Chlo ide Ion
Concen a ion [online] [ iewed 6 Oc 2016]. A ailable om:
ww2.chemis y.ga ech.edu/class/analy / ia.pd
DE LAMO, D., 2014. Diseño y cons ucción del p o o ipo de un sis ema de Análisis de
Inyección Secuencial pa a la moni o ización de p ocesos median e lenguas
elec ónicas
LARSEN, D. and D. HARVEY, 2013. Flow Injec ion Analysis [online] [ iewed 6 Dec 2016].
A ailable om:
h p://chemwiki.ucda is.edu/Co e/Analy ical_Chemis y/Analy ical_Chemis y_2.0/13_Ki
ne ic_Me hods/13.4:_Flow_Injec ion_Analysis
MACDOUGALL, D. and W.B. CRUMMETT, 1980. Guidelines o Da a Acquisi ion and Da a
Quali y E alua ion in En i onmen al Chemis y. Analy ical Chemis y [online], 52(14),
2242–2249 A ailable om: <Go o ISI>://WOS:A1980KT61200006
DEL MAR, B. i L.M., 2004. Nue as Es a egias Pa a La Ges ión de Fluidos En Sis emas
Au oma izados de Análisis. Uni e si a Au ònoma de Ba celona. Uni e si a Au ònoma
de Ba celona
NIČ, M. e al., eds., 2009. IUPAC Compendium o Chemical Te minology [online]. Resea ch
T iagle Pa k, NC: IUPAC [ iewed 2 Jun 2016]. A ailable om: h p://goldbook.iupac.o g
NÚÑEZ, J.L., 2016. Es udio de la luidica asociada a la op imización de un sis ema de
análisis po inyección secuencial (SIA)
Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 69
NUÑEZ, L. e al., 2013. De elopmen and applica ion o an elec onic ongue o de ec ion
and moni o ing o ni a e, ni i e and ammonium le els in wa e s. Mic ochemical Jou nal
[online], 110, 273–279 A ailable om: h p://dx.doi.o g/10.1016/j.mic oc.2013.04.018
PASEKOVA, H., M. POLASEK and P. SOLICH, 1999. Sequen ial injec ion analysis.
Chemické lis y, 93(6), 354–359
PINTO, P.C.A.G. e al., 2011. Sequen ial Injec ion Analysis Hyphena ed wi h O he Flow
Techniques: A Re iew. Analy ical Le e s, 44(1-3), 374–397
ROBINSON, J.W., E.M. SKELLY FRAME and G.M. FRAME II, 2005. Unde g adua e
Ins umen al analysis, 1, 1079
SOCHACKA, J., 2015. Applica ion o phenol ed as a ma ke ligand o bili ubin binding si e
a subdomain IIA on human se um albumin. Jou nal o pho ochemis y and
pho obiology. B, Biology [online], 151, 89–99 A ailable om:
h p://www.ncbi.nlm.nih.go /pubmed/26231934
VINDEVOGHEL, W., 2005. Modi ica ion o a SIA-sys em by addi ion o a s i e de ice
(mixing low cell)
Addi ional bibliog aphic e e ences
AVE, D., 2015. Flame Minia u e Spec ome e Use Manual
BAXTER, P.J. and G.D. CHRISTIAN, 1996. Sequen ial Injec ion Analysis: A Ve sa ile
Technique o Biop ocess Moni o ing. Accoun s o Chemical Resea ch [online], 29(11),
515–521 A ailable om: h p://pubs.acs.o g/doi/abs/10.1021/a 950214z
CHRISTIAN, G.D., 2003. Flow analysis and i s ole and impo ance in he analy ical
sciences. Analy ica Chimica Ac a, 499(1-2), 5–8
ESCUDÉ, B., 2015. (SIA), Op imi zació d’un sis ema de moni o i zació de p ocessos basa
en anàlisi pe injecció seqüencial
FIALAB, 2015. Fibe Op ic SMA Z-Flow Cell Manual Design
GUBELI, T., G.D. CHRISTIAN and J. RUZICKA, 1991. Fundamen als o Sinusoidal Flow
Sequen ial Injec ion Spec opho ome y. Analy ical Chemis y, 63, 2407–2413
HARVEY, D., 2009. Analy ical Chemis y 2.0, 810
KIKAS, T., 2014. In oduc ion o Flow Injec ion Analysis ( FIA ) De e mina ion o Chlo ide Ion
Concen a ion [online] [ iewed 6 Oc 2016]. A ailable om:
ww2.chemis y.ga ech.edu/class/analy / ia.pd
DE LAMO, D., 2014. Diseño y cons ucción del p o o ipo de un sis ema de Análisis de
Inyección Secuencial pa a la moni o ización de p ocesos median e lenguas
elec ónicas
LARSEN, D. and D. HARVEY, 2013. Flow Injec ion Analysis [online] [ iewed 6 Dec 2016].

p. 70 Repo
A ailable om:
h p://chemwiki.ucda is.edu/Co e/Analy ical_Chemis y/Analy ical_Chemis y_2.0/13_Ki
ne ic_Me hods/13.4:_Flow_Injec ion_Analysis
MACDOUGALL, D. and W.B. CRUMMETT, 1980. Guidelines o Da a Acquisi ion and Da a
Quali y E alua ion in En i onmen al Chemis y. Analy ical Chemis y [online], 52(14),
2242–2249 A ailable om: <Go o ISI>://WOS:A1980KT61200006
DEL MAR, B. i L.M., 2004. Nue as Es a egias Pa a La Ges ión de Fluidos En Sis emas
Au oma izados de Análisis. Uni e si a Au ònoma de Ba celona. Uni e si a Au ònoma
de Ba celona
NIČ, M. e al., eds., 2009. IUPAC Compendium o Chemical Te minology [online]. Resea ch
T iagle Pa k, NC: IUPAC [ iewed 2 Jun 2016]. A ailable om: h p://goldbook.iupac.o g
NÚÑEZ, J.L., 2016. Es udio de la luidica asociada a la op imización de un sis ema de
análisis po inyección secuencial (SIA)
NUÑEZ, L. e al., 2013. De elopmen and applica ion o an elec onic ongue o de ec ion
and moni o ing o ni a e, ni i e and ammonium le els in wa e s. Mic ochemical Jou nal
[online], 110, 273–279 A ailable om: h p://dx.doi.o g/10.1016/j.mic oc.2013.04.018
PASEKOVA, H., M. POLASEK and P. SOLICH, 1999. Sequen ial injec ion analysis.
Chemické lis y, 93(6), 354–359
PINTO, P.C.A.G. e al., 2011. Sequen ial Injec ion Analysis Hyphena ed wi h O he Flow
Techniques: A Re iew. Analy ical Le e s, 44(1-3), 374–397
ROBINSON, J.W., E.M. SKELLY FRAME and G.M. FRAME II, 2005. Unde g adua e
Ins umen al analysis, 1, 1079
SOCHACKA, J., 2015. Applica ion o phenol ed as a ma ke ligand o bili ubin binding si e
a subdomain IIA on human se um albumin. Jou nal o pho ochemis y and
pho obiology. B, Biology [online], 151, 89–99 A ailable om:
h p://www.ncbi.nlm.nih.go /pubmed/26231934
VINDEVOGHEL, W., 2005. Modi ica ion o a SIA-sys em by addi ion o a s i e de ice
(mixing low cell)
Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 71
Table o igu es
Figu e 4-1 Fou phases o Flow Injec ion (Kikas 2014) ........................................................ 12
Figu e 4-2 S eam di usion .................................................................................................. 13
Figu e 4-3 Basic FIA mani old (La sen and Ha ey 2013) .................................................... 13
Figu e 4-4 Con igu a ion o a basic SIA sys em. C: ca ie , PP: pumping de ice; SV: selec ion
al e; HC: holding coil; RC: eac ion coil; D: de ec o ; W: was e; R: eagen ; S: sample (Pin o
e al. 2011) ........................................................................................................................... 14
Figu e 5-1 Elec omagne ic Wa e o m (Ha ey 2009) ......................................................... 15
Figu e 5-2 Elec omagne ic spec um (Ha ey 2009) ........................................................... 16
Figu e 5-3 Bee - Lambe law ac o s modi ied om (Ha ey 2009) .................................... 17
Figu e 5-4 Flame-S de ec o o e iew (A e 2015) ............................................................... 19
Figu e 5-5 Le : Non a e aged spec a. Righ : 15 scans a e aged ....................................... 19
Figu e 6-1 SIA schema ic modi ied om (Núñez 2016) ........................................................ 21
Figu e 6-2 SIA assembly ...................................................................................................... 22
Figu e 6-3 Mul iBu e e 2S .................................................................................................... 22
Figu e 6-4 SIA on iew ...................................................................................................... 24
Figu e 6-5 Val e posi ions ( on iew) ................................................................................. 24
Figu e 6-6 Mixing Cell........................................................................................................... 25
Figu e 6-7 Sc ip File B owse .............................................................................................. 28
Figu e 6-8 Sc ip execu ion ................................................................................................... 29
Figu e 7-1 Spec oscopy sys em .......................................................................................... 30
Figu e 7-2 Flame S Spec ome e ........................................................................................ 30
Figu e 7-3 SMA-Z-10-UL ...................................................................................................... 31
Figu e 7-4 DH-mini UV-VIS-NIR Ligh sou ce ....................................................................... 32
p. 72 Repo
Figu e 7-5 Spec oscopy applica ion wiza d ......................................................................... 32
Figu e 7-6 Concen a ion Choice .......................................................................................... 33
Figu e 7-7 Se Aquisi ion Pa ame e s ................................................................................... 34
Figu e 7-8 S o e Re e ence and Backg ound spec um ....................................................... 34
Figu e 7-9 Wa eleng h selec ion .......................................................................................... 35
Figu e 7-10 Da a sa ing con igu a ion sc een ...................................................................... 36
Figu e 8-1 Phenol ed in: Le : basic medium Righ : acid medium ........................................ 37
Figu e 8-2 Phenol ed abso p ion spec um (Sochacka 2015) ............................................. 38
Figu e 8-3 Reagen aspi a ion p ocedu e modi ied om (Núñez 2016) ................................ 38
Figu e 8-4 S anda d en ies .................................................................................................. 39
Figu e 8-5 Regions o de ec ion and quan iza ion (MacDougall and C umme 1980) .......... 41
Figu e 8-6 Theo e ical cu es o Dispe sion coe icien s olume(Gubeli e al. 1991) ........ 42
Figu e 9-1 Phenol ed p elimina y abso p ion spec um ....................................................... 43
Figu e 9-2 Le : co e ed cell. Cen e : Unco e ed cell, ligh s o . Righ : Unco e ed cell ........ 44
Figu e 9-3 Re e ence and backg ound spec ums: Top: Unco e ed cell. Bo om: co e ed cell
............................................................................................................................................. 44
Figu e 9-4 Abso p ion peaks when he simple cell is unco e ed .......................................... 45
Figu e 9-5 Phenol ed spec um o 2, 10, 40 ppm ............................................................... 46
Figu e 9-6 Le : Calib a ion cu e o Flame spec ome e a 550 and 560 nm. Righ :
Calib a ion a ixed wa eleng h (560 nm) wi h Flame and UV-Mini 1240 spec ome e s ...... 46
Figu e 9-7 Sample decay o e ime ...................................................................................... 47
Figu e 9-8 S anda ds' abso bance spec um ........................................................................ 47
Figu e 9-9 O iginal s anda ds’ calib a ion cu e ................................................................... 48
Figu e 9-10 Mul i al e p iming ............................................................................................. 49
Implemen a ion and op imiza ion o a sequen ial injec ion analysis (SIA) sys em by UV-Visible spec oscopy p. 73
Figu e 9-11 Abso bance o di e en injec ed olume .......................................................... 50
Figu e 9-12 Abso bance a io and dispe sion coe icien ...................................................... 50
Figu e 9-13 Calib a ion cu es Le : 1-20 ppm Righ : 1-16 ppm ........................................... 51
Figu e 9-14 Calib a ion 2016-6-1 .......................................................................................... 52
Figu e 9-15 Con idence in e als o abso p i i y o e ime .................................................. 53
Figu e 9-16 Peak cu es compa ison ................................................................................... 54
Figu e 9-17 Upwa ds and downwa ds calib a ions ............................................................... 55
Figu e 9-18 Ex e nal calib a ion and 50 % dilu ions Mul i al e po s a e labelled M1, M2, and
M3 Le : Ascending o de Righ : Descending o de .............................................................. 56
Figu e 9-19 In e nal calib a ions using 20 ppm as s ock solu ion .......................................... 57
Figu e 9-20 Calib a ion om s ock solu ion .......................................................................... 58
Figu e 9-21 Successi e dilu ion cu es ................................................................................ 59
Figu e 9-22 Cleaning spec al da a....................................................................................... 60