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NUEVAS METODOLOGÍAS ANALÍTICAS

PARA LA DETERMINACIÓN DE
ANTIOXIDANTES ALIMENTARIOS


NEW ANALYTICAL METHODOLOGIES
FOR FOOD ANTIOXIDANT
DETERMINATION





Tesis Doctoral
Álvaro Andreu Navarro
Noviembre 2011
TÍTULO:Nuevas metodologías analíticas para la determinación de
antioxidantes alimentarios
AUTOR: Álvaro Andreu Navarro
© Edita: Servicio de Publicaciones de la Universidad de Córdoba. 2012
Campus de Rabanales
Ctra. Nacional IV, Km. 396 A
14071 Córdoba
www.uco.es/publicaciones
publicaciones@uco.es


!








TÍTULO DE LA TESIS: NUEVAS METODOLOGÍAS ANALÍTICAS PARA LA
DETERMINACIÓN DE ANTIOXIDANTES ALIMENTARIOS


DOCTORANDO/A: ÁLVARO ANDREU NAVARRO


INFORME RAZONADO DEL/DE LOS DIRECTOR/ES DE LA TESIS
(se hará mención a la evolución y desarrollo de la tesis, así como a trabajos y publicaciones
derivados de la misma).

El uoctoianuo B. Alvaio Anuieu Navaiio cuiso
biillantemente los estuuios uel Nastei en Quimica Fina Avanzaua,
obtenienuo excelentes calificaciones en las asignatuias uel mismo. El
tiabajo fin ue Nástei se publico en la ievista }ouinal of Agiicultuial
anu Foou Chemistiy, ue iefeiencia en el áiea ue conocimiento.

La temática ue la Tesis se encuauia uentio ue las tenuencias
ue la Quimica Analitica ya que aboiua nuevas metouologias analiticas
paia la ueteiminacion ue antioxiuantes alimentaiios meuiante
sistemas automáticos ue análisis ciomatogiáficos y no
ciomatogiáficos. Se han estableciuo nuevos métouos paia la
ueteiminacion ue polifenoles meuiante sepaiacion ciomatogiáfica y
ueiivatizacion post-columna, junto con uetectoies fotométiicos y
fluoiimétiicos. También se ha uesaiiollauo un métouo quimiométiico
paia la clasificacion ue vinos utilizanuo la infoimacion obteniua en
uno ue los métouos ciomatogiáficos piopuestos. En la segunua paite
ue la Tesis se uesciiben uos métouos no ciomatogiáficos basauos en
el uso ue la técnica ue mezcla ue flujo ueteniuo y ue nanopaiticulas ue
oio, lo que ha peimitiuo ampliai el campo ue aplicacion analitica ue
estas nanopaiticulas. Es ue uestacai que se ha uemostiauo la utiliuau
piáctica ue touos los métouos uesaiiollauos meuiante su aplicacion al
análisis ue uistintos tipos ue alimentos.






Neuiante la uefensa ue esta Nemoiia se pietenue optai a
la obtencion ue la mencion "Boctoiauo Euiopeo" habiua cuenta
ue que el uoctoianuo ieúne los iequisitos exigiuos paia tal
mencion:


1. Cuenta con los infoimes favoiables ue uos uoctoies
peitenecientes a Instituciones ue Enseñanza Supeiioi ue
paises euiopeos uistintos a España.

2. 0no ue los miembios uel tiibunal que ha ue evaluai la
Tesis peitenece a un centio ue enseñanza supeiioi ue
otio pais euiopeo.

S. Paite ue la uefensa ue la Nemoiia se iealizaiá en la
lengua oficial ue otio pais euiopeo.

4. El uoctoianuo ha iealizauo una estancia en el Institut füi
Analytische Chemie, Chemo- unu Biosensoiik ue la
0niveisiuau ue Regensbuig, Alemania, ue ties meses ue
uuiacion, que ha contiibuiuo a su foimacion y peimitiuo
uesaiiollai paite uel tiabajo expeiimental ue esta
Nemoiia.




Agiauezco a la Consejeiia ue Innovacion, Ciencia y Empiesa
ue la }unta ue Anualucia la concesion ue una beca pieuoctoial
ausciita al Pioyecto ue Excelencia Pu6-FQN-u1SS6 que me ha
peimitiuo ueuicai estos últimos 4 años al uesaiiollo ue esta Tesis.






































































uiacias a touos aquellos que
me han uiiigiuo, ayuuauo,
apoyauo y acompañauo a lo
laigo ue esta Tesis Boctoial
















































































A mi mauie, poique giacias a
su esfueizo me ha uauo la
posibiliuau ue llegai hasta aqui



















































ÍNDICE





























ÍNDICE Pág

Objeto / Aim 1

Introducción 7

Capítulo 1: Herramientas Analíticas S1

1. Estánuaies y ieactivos

S4
2. Sistemas FIA S6
S. Instiumentacion S7
4. Apaiatos S7
S. Piogiamas infoimáticos S8

Capítulo 2: Innovaciones en la determinación
cromatográfica de antioxidantes en alimentos S9

Intiouuccion

61
1. Analytical innovations in the uetection of
phenolics in wines 6S
2. 0sefulness of teibium-sensitizeu luminiscence
uetection foi the chemometiic
classification of wines by theii content in
phenolic compounus. 91
S. Luminescent ueteimination of flavonoius in
oiange juices by liquiu chiomatogiaphy with
post-column ueiivatization with aluminium
anu teibium 11S
4. Long-wavelenght fluoiescence uetection of
flavonoius in oiange juices using liquiu
chiomatogiaphy 1SS

Capítulo 3: Innovaciones en la determinación no
cromatográfica de antioxidantes en alimentos 1S1

Intiouuccion 1SS
1. Beteimination of antioxiuant auuitives in
fooustuffs by uiiect measuiement of golu
nanopaiticle foimation using iesonance light
scatteiing uetection 1SS
2. Beteimination of polyphenolic content in
beveiages using laccase, golu nanopaiticles
anu long wavelenght fluoiimetiy 17S
Capítulo 4: Nuevas investigaciones con nanopartículas de
oro
192

Intiouuccion

194
1. 0bjetivos 194
2. Conocimientos auquiiiuos 194
S.Tiabajo Expeiimental

2u2
Capítulo 5: Discusión de resultados 2u8

Intiouuccion

21u
1. Netouologias basauas en sepaiacion
ciomatogiáfica, ueiivatizacion post
columna y ueteccion fluoiescente
21u
2. Netouologias automáticas no ciomatogiáficas
basauas en el uso ue la técnica ue flujo
ueteniuo y nanopaiticulas.
222
Discussion of the results 227

Intiouuction

227
1. Nethouologies baseu on chiomatogiaphic
sepaiation, post-column ueiivatization anu
fluoiescence uetection 227
2. Automatic non-chiomatogiaphic
methouologies baseu in the useu of stoppeu-
flow technique anu nanopaiticles 2S8

Conclusiones / Conclusions 24S

Anexo 249













OBJETO
AIM





































S
OBJETO



El objetivo genéiico ue las investigaciones que se iecogen en esta
Nemoiia ha siuo el uesaiiollo ue nuevas metouologias analiticas
ciomatogiáficas y no ciomatogiáficas que contiibuyan a mejoiai el contiol ue
la caliuau alimentaiia. Los estuuios iealizauos se han centiauo en la
ueteiminacion ue antioxiuantes natuiales y sintéticos en alimentos. Paia
alcanzai este objetivo se han iealizauo las investigaciones siguientes:

- Estuuio ue la aplicabiliuau ue nuevas ieacciones ue
ueiivatizacion postcolumna en ciomatogiafia ue liquiuos
utilizanuo la luminiscencia sensibilizaua ue teibio(III) y la
fluoiescencia ue laiga longituu ue onua como sistemas ue
ueteccion paia la ueteiminacion ue compuestos fenolicos.

- 0tilizacion ue métouos quimiométiicos en análisis
clasificatoiio meuiante ciomatogiafia ue liquiuos y ueteccion
fotométiica y fluoiimétiica.

- Investigacion ue la capaciuau ue uiveisos auitivos
antioxiuantes alimentaiios paia oiiginai nanopaiticulas ue
oio y su aplicacion paia la ueteiminacion ue estos auitivos
utilizanuo la uispeision ue la iauiacion como sistema ue
ueteccion.

- Estuuio ue la utiliuau uel uso conjunto ue la fluoiimetiia ue
laiga longituu ue onua, la oxiuacion enzimática y
nanopaiticulas ue oio paia la ueteiminacion uel conteniuo ue
polifenoles en alimentos.

- Aplicacion ue los métouos piopuestos al análisis ue alimentos
con objeto ue uemostiai su utiliuau piáctica.
S
AIM



The geneial aim of the ieseaich incluueu in this stuuy has been the
uevelopment of new analytical chiomatogiaphic anu non chiomatogiaphic
methouologies that contiibute to impiove the contiol in foou quality. The
ieseaiches have focuseu on the ueteimination of natuial anu synthetic
antioxiuants in foous. The following investigations have been uevelopeu to
achieve this goal:

- Stuuy of the applicability of new post-column ueiivatization
ieactions in liquiu chiomatogiaphy using teibium(III)
sensitizeu luminescence anu long wavelength fluoiescence as
uetection systems.

- 0se of chemometiic methous in classification analysis by liquiu
chiomatogiaphy using photometiic anu fluoiimetiic uetection.

- Investigation of the ability of seveial foou antioxiuant auuitives
to foim golu nanopaiticles anu its application to the
ueteimination of these auuitives using iesonance light
scatteiing as uetection system.

- Stuuy of the usefulness of the combineu use of long wavelength
fluoiimetiy, enzymatic oxiuation anu golu nanopaiticles foi the
ueteimination of polyphenols in foous.

- Application of the pioposeu methous in foou analysis to
uemonstiate theii piactical usefulness.


















INTRODUCCIÓN






































































Intiouuccion


9
En esta intiouuccion se comentaián algunos aspectos básicos
ielacionauos con las especies quimicas que se han utilizauo como analitos
en las investigaciones iealizauas y las piincipales heiiamientas
instiumentales y metouologicas que han peimitiuo el uesaiiollo ue los
métouos ueteiminativos piesentauos. Puesto que la Nemoiia está ueuicaua
al establecimiento ue nuevos métouos paia la ueteiminacion ue
antioxiuantes alimentaiios, natuiales y sintéticos, se uesciiben bievemente
las piincipales piopieuaues y el mouo ue actuacion ue los compuestos más
iepiesentativos ue este giupo, el inteiés poi contiolai su piesencia en los
alimentos y algunas caiacteiisticas ue los métouos ueteiminativos
pieviamente uesciitos. En lo iefeiente a las heiiamientas utilizauas, se
comentan sucintamente los anteceuentes más uestacables sobie la
aplicabiliuau analitica ue la luminiscencia sensibilizaua ue lantániuos, la
fluoiescencia ue laiga longituu ue onua y la uispeision ue la iauiacion como
sistemas ue ueteccion. También se incluyen algunos aspectos actuales
sobie análisis clasificatoiio y sistemas ue flujo.


ANTIOXIDANTES ALIMENTARIOS

Los antioxiuantes alimentaiios son compuestos quimicos que en
pequeñas cantiuaues evitan o ietiasan la oxiuacion ue sustancias
fácilmente oxiuables, tales como los lipiuos |1j. La oxiuacion ue lipiuos en
los alimentos es un pioceso complejo en el que fiecuentemente inteivienen
iauicales libies y que pueue piouuciise uuiante la iecoleccion,
piocesamiento y almacenamiento ue los alimentos |2j. Factoies como caloi,
luz, oxigeno, enzimas, metales ue tiansicion, metalopioteinas y
miciooiganismos pueuen actuai como iniciauoies o piomotoies ue este
pioceso uanuo lugai a alteiaciones en el aioma y saboi uel alimento, a la
péiuiua ue áciuos giasos esenciales, ue vitaminas liposolubles y ue otios
compuestos bioactivos, y a la foimacion ue especies potencialmente
toxicas.

Existen uiveisos compuestos que se encuentian ue foima natuial en
los alimentos y que piesentan piopieuaues antioxiuantes, piotegienuo a los
tejiuos ue piocesos oxiuativos a tiavés ue la uieta. Los piincipales
antioxiuantes natuiales son los polifenoles (flavonoiues y áciuos
polifenolicos) junto con vitamina C (áciuo ascoibico), vitamina E

1u
(tocofeioles) y caiotenoiues |Sj. Algunos ue estos compuestos se utilizan
como auitivos alimentaiios aunque, uebiuo a su escasa estabiliuau, la
inuustiia alimentaiia también utiliza uesue los años cuaienta antioxiuantes
sintéticos que son compuestos fenolicos, tales como butilhiuioxianisol
(BBA), butilhiuioxitolueno (BBT) y galato ue piopilo (uP). No obstante, su
uso actualmente está iestiingiuo a cieitos alimentos y en cantiuaues
limitauas, uebiuo piincipalmente, a sus potenciales efectos canceiigenos.

El mecanismo ue actuacion y la eficacia ue los antioxiuantes
uepenuen ue sus caiacteiisticas fisicas y quimicas y uel sistema implicauo
|2j. Estas sustancias pueuen actuai a uistintos niveles en el pioceso
oxiuativo ue los lipiuos: 1) evitanuo la foimacion ue iauicales o
inteiaccionanuo con ellos, 2) actuanuo como complejantes ue iones
metálicos como Fe(II) y Fe(III), lo que impiue su efecto catalitico en la
foimacion ue lipopeioxiuos, S) inhibienuo a enzimas piooxiuativas, 4)
uescomponienuo los peioxiuos y otias especies con oxigeno ieactivo (R0S,
ieactive oxygen species), y S) inteifiiienuo ieacciones en las que están
implicauos iauicales peioxilo (L00
·
) o alcoxilo (L0
·
).

La evaluacion ue la capaciuau y la activiuau antioxiuante ue un
compuesto es un pioceso complejo ya que ueben estuuiaise los posibles
mecanismos ue actuacion en uifeientes conuiciones, tenienuo en cuenta las
piopieuaues multifuncionales que pueue piesentai |1j. A veces, al iealizai
investigaciones ue este tipo se han obteniuo iesultauos contiauictoiios, lo
que se ha atiibuiuo a factoies tales como caiacteiisticas uel ensayo, la
natuialeza uel sustiato utilizauo y la influencia ue otios componentes uel
sistema. Bay que tenei en cuenta que el estuuio ue estos factoies utilizanuo
como mouelo un sistema simple pueue sei sencillo, peio la situacion es más
pioblemática en un sistema heteiogéneo como es la matiiz ue un alimento
o el oiganismo humano. 0n métouo simple paia evaluai la eficacia ue un
antioxiuante es meuiante un ensayo compaiativo, utilizanuo el uenominauo
"factoi antioxiuativo" (AF): I
A
¡I
0
, uonue I
A
e I
0
son los peiiouos ue
inuuccion obteniuos paia una giasa o aceite en piesencia y ausencia,
iespectivamente, ue un antioxiuante. El valoi ue AF, poi tanto, es mayoi
cuanto más eficaz es el antioxiuante.

El uesaiiollo ue métouos paia la ueteiminacion ue antioxiuantes en
alimentos es una linea ue investigacion bastante activa, como se muestia en
Intiouuccion


11
la Figura 1, uonue pueue obseivaise el ciecimiento exponencial ue las
publicaciones sobie esta temática en los últimos años. A continuacion se
uesciiben bievemente las caiacteiisticas más uestacables ue estos
compuestos y los piincipales métouos uesciitos paia su ueteiminacion.
















Figura 1. Bistiibucion a lo laigo uel tiempo ue las publicaciones ielacionauas
con métouos paia la ueteiminacion ue antioxiuantes. (Base ue uatos
SC0P0S).


1. Antioxidantes naturales

0no ue los giupos más iepiesentativos ue los antioxiuantes
natuiales lo constituyen los compuestos fenolicos, al que peitenecen los
flavonoiues y los áciuos fenolicos junto con taninos, estilbenos y lignanos
|4j. Estos compuestos son metabolitos secunuaiios ue las plantas y se
consiueian iesponsables ue la caliuau sensoiial y nutiicional ue fiutas y
veiuuias, uonue se encuentian ampliamente uistiibuiuos, asi como aceite
ue oliva y te, entie otios alimentos. La potencial toxiciuau ue los
antioxiuantes sintéticos ha uauo lugai a que aumente el inteiés poi estos
compuestos ue oiigen natuial, a los que también se les atiibuyen
piopieuaues antimiciobianas, antiviiales y antiinflamatoiias.

En la Figura 2 se muestian las estiuctuias ue los piincipales
componentes ue los polifenoles, ue los que se han uesciito más ue 8.uuu

12




































Figura 2. Estiuctuias quimicas ue los piincipales clases ue compuestos
fenolicos.
R
1

R
2

R
3

R
1
=R
2
=R
3
=OH:Ácidogálico
R
1
=R
2
=OH,R
3
=-:Ácidoprotocatecuico
Flavonoides Ácidos hidroxibenzoicos
Gallotaninas
(ej. pentagalloyglucosa)
R
1

R
2

Ácidos hidroxicinámicos
R
1
=OH: Ácido cumárico
R
1
=R
2
=OH: äcido cafeico
Estilbenos (ej. Resveratrol)
Intiouuccion


1S

Figura 2. Continuacion



compuestos. 0no ue los subgiupos más numeiosos lo constituyen los
flavonoiues, los cuales pueuen encontiaise bien en foima libie o como
glucosiuos. Existen más ue 4uuu flavonoiues, ue los que unos Suu se
encuentian en foima libie |Sj. El esqueleto ue los flavonoiues está foimauo
poi ties anillos fenolicos uenominauos A, B y C (Figura 3). El anillo A está
conuensauo con el anillo C, el cual tiene uniuo en la posicion 2 al anillo B
como sustituyente. Se uiviuen en vaiias clases según su estauo oxiuativo y
sus sustituyentes. Como muestia la Figura 3, el anillo C pueue sei un
piiano heteiociclico (antocianiuinas y catequinas o flavanoles) o una piiona
(flavonas, flavonoles, flavononas e isoflavonas). A los flavonoiues ue este
último giupo, los cuales tienen un giupo caibonilo en el C-4 uel anillo C, se
les uenomina fiecuentemente 4-oxo-flavonoiues.

Los áciuos fenolicos constituyen, junto con los flavonoiues otio
giupo impoitante ue compuestos fenolicos pioceuentes piincipalmente ue
las plantas |6j. Las estiuctuias ue los piincipales componentes ue este
giupo se muestian en la Tabla 1. Entie ellos se encuentian los que tienen
la estiuctuia uel áciuo hiuioxicinamico, como los áciuos cafeico, p-
cumáiico y feiúlico, y los que tienen la estiuctuia uel áciuo
hiuioxibenzoico, como los áciuos vainillico, piotocatecuico, gentisico y
R
1

R
2

R
2

R
2

Proantocianidinas (R
1
, R
2
=H, OH)
(ej. Dímero de procianidina tipo-B,
R
1
=OH, R
2
=H)
Lignanos
(ej. Secoisolaiiciiesinol)

14
siiingico . Estos compuestos iealizan uiveisas funciones en la viua ue las
plantas y afectan a sus caiacteiisticas sensoiiales.
























Figura 3. Estiuctuias quimicas ue las piincipales clases ue flavonoiues.

Los polifenoles tienen numeiosas aplicaciones inuustiiales ya que
se utilizan como conseivantes y coloiantes natuiales en alimentos y en la
piouuccion ue pintuias, papel y cosméticos. Actualmente tienen un gian
inteiés los piocesos ue tiatamiento ue iesiuuos ue inuustiias alimenticias
ya que éstos pueuen contenei cantiuaues impoitantes ue compuestos
fenolicos. Poi ejemplo en los iesiuuos ue las extiactoias ue aceitunas existe
entie el 1 y el 1,8 % ue conteniuo fenolico, según la vaiieuau ue aceituna
|7j. También se han uesciito las hojas uel olivo como fuente ue estos
compuestos |8j y los uesechos ue la inuustiia ue los citiicos y ue otias
fiutas |9j.
CATEQUINAS ANTOCIANINAS
FLAVONAS FLAVONOIDES
FLAVANONAS ISOFLAVONAS
Intiouuccion


1S


Tabla 1. Estiuctuias ue los áciuos fenolicos más abunuantes ue oiigen
natuial











R
2
R
S
R
4
R
S
X Nombie Común
B B B B a áciuo cinámico
-0B B B B a áciuo o-cumáiico
B B -0B B a áciuo p-cumáiico
B -0B B B a áciuo m-cumáiico
B -0CB
S
-0B B a áciuo feiúlico
B -0CB
S
-0B -0CB
S
a áciuo sinápico
B -0B -0B B a áciuo cafeico
B B B B b áciuo benzoico
-0B B B B b áciuo salicilico
B B -0B B b áciuo p-hiuioxibenzoico
B -0CB
S
-0B B b áciuo vainillico
B -0CB
S
-0B -0CB
S
b áciuo siiingico
B -0B -0B B b áciuo piotocatecuico
-0B B B -0B b áciuo gentisico
-0B -0B -0B -0B b áciuo gálico
B -0CB
S
-0CB
S
B b áciuo veiátiico
B -0CB
S
-0B -0CB
S
c siiingaluehiuo
B -0CB
S
-0B B c vainillina


Entie las piincipales fuentes ue compuestos fenolicos en la uieta
humana se encuentian los vinos, las bebiuas ue zumo ue fiutas y el té. En la
Tabla 2 se muestia el conteniuo ue polifenoles totales en algunas ue estas
bebiuas. El vino contiene una gian vaiieuau ue flavonoiues (flavonol,
flavan-S-ol y antocianinas) y no flavonoiues (áciuos fenolicos, alcoholes
fenolicos, estilbeno y áciuo hiuioxicinnamico) |1uj. El peifil ue polifenoles
vaiia ue los vinos tintos a los blancos uebiuo a la vaiieuau ue uva y al
pioceso ue vinificacion. El elevauo conteniuo en polifenoles ue los vinos

16
tintos ha uauo lugai a que se les consiueie más piotectoies ue la saluu que
otias bebiuas alcoholicas |11j.


Tabla2. Conteniuo apioximauo ue compuestos fenolicos en bebiuas

Tipo ue bebiua Conteniuo apioximauo ue compuestos fenolicos
zumos comeiciales
Nanzana SS9± 4S
a
0va SSS ± 11
a
Naianja 7SS ± 18
a
Piña SS8 ± S
a
Zumos natuiales
0va (ioja) 1728
a
0va (blanca) S19
a
Tés
Te negio 8u.S - 1S4.9
b
Te veiue 6S.8 - 1u6.2
b
Te veiue 61 - 2uu
b
vinos tintos
Aigentino 1S9S - 16S7
a
Biasileño 1947 - 1984
a
Español 1869
a
Fiancés 1847 - 26uu
a
vinos blancos
Aigentino 216
a
Biasileño 2S6 - SSS
a
Fiancés 24S
a
Español 292
a
a
mg L
-1
;
b
mg g
-1
. Refeiiuo a áciuo gálico

Existe una bibliogiafia muy extensa sobie los efectos beneficiosos
ue los compuestos fenolicos en la saluu |1,2,4,S,12,1Sj. Estuuios
epiuemiologicos han puesto ue manifiesto que la uieta iica en polifenoles se
coiielaciona con el aumento ue la longeviuau y la uisminucion en la
inciuencia ue las enfeimeuaues caiuiovasculaies en la poblacion, aun
cuanuo la ingestion ue giasas sea elevaua |14j. Auemás ue las piopieuaues
antioxiuantes ue los polifenoles, se ha uemostiauo que estos compuestos
piesentan numeiosas piopieuaues, actuanuo como antiviiales, anti-
bacteiianos, anti-inflamatoiios, vasouilatauoies, anti-canceiigenos y anti-
isquémicos. También inhiben la foimacion ue lipopeioxiuos y la agiegacion
plaquetaiia y mejoian la peimeabiliuau y la fiagiliuau capilai.

Intiouuccion


17
Los polifenoles pievienen el uaño ue los componentes celulaies
piouuciuo poi ieacciones quimicas en las que están implicauos iauicales
libies. El oxigeno existente en el oiganismo es una fuente impoitante ue
iauicales libies, tales como los iauicales hiuioxilo (
·
0B) e hiuiopeioxilo
(B0
2
·
), el anion supeioxiuo (0
2
·-
), y ue peioxiuo ue hiuiogeno |1Sj. Estas
especies pueuen inteiaccionai con lipiuos, enzimas y otias pioteinas y
áciuos nucleicos, sienuo la oxiuacion ue áciuos giasos insatuiauos
piesentes en las membianas ue las células el pioceso oxiuativo más
fiecuente en el oiganismo. Los iauicales libies ue oxigeno foimauos
uuiante el metabolismo pueuen alcanzai niveles citotoxicos uañanuo las
membianas ue las células, piovocanuo su iuptuia, y a otios componentes
celulaies o extiacelulaies tales como enzimas, lipopioteinas y otias
pioteinas, colesteiol y mateiial genético uel núcleo. Touo ello pueue uai
lugai a una amplia vaiieuau ue patologias, tales como piocesos
inflamatoiios, aiteiioescleiosis, caicinogénesis y envejecimiento, entie
otias.

También hay que inuicai que, a pesai ue sus efectos beneficiosos,
los polifenoles pueuen actuai en cieitas ciicunstancias como piooxiuantes,
al igual que otios antioxiuantes, favoiecienuo la oxiuacion ue cieitos
compuestos. Poi ejemplo, la activiuau piooxiuante ue los flavonoiues es
uiiectamente piopoicional al númeio ue giupos hiuioxilo ya que se ha
uemostiauo que los mono- y uihiuioxiflavonoiues no piesentan esta
piopieuau ue foima uetectable, mientias que la piesencia ue múltiples
giupos hiuioxilo, especialmente en el anillo B, aumenta significativamente
la piouuccion ue iauicales hiuioxilo a tiavés ue la ieaccion ue Fenton |14,
16,17j. En geneial, existe un consenso sobie la necesiuau ue piofunuizai en
el efecto ue los polifenoles en el oiganismo humano uebiuo a que se han
uauo opiniones contiauictoiias |17j.

El inteiés que existe poi la ielacion entie los compuestos fenolicos
y las piopieuaues oiganolépticas, antioxiuantes y nutiicionales ue los
alimentos, ha uauo lugai al uesaiiollo ue una gian vaiieuau ue métouos
analiticos en los que se incluyen las coiiesponuientes etapas ue extiaccion,
sepaiacion y cuantificacion |4j. La extiaccion ue estos compuestos ue la
matiiz ue la muestia es una etapa ciitica, aunque no existe un métouo
estánuai con este fin. Las uos técnicas más utilizauas son la extiaccion con
uisolventes oigánicos y la extiaccion con fluiuos supeiciiticos, aunque en

18
ambos casos se piouuce la co-extiaccion ue otios compuestos tales como
azúcaies, áciuos oigánicos y pioteinas, poi lo que se necesitan piocesos
posteiioies ue puiificacion como, poi ejemplo, extiaccion en fase soliua.

La extiaccion con uisolventes pueue sei liquiuo-liquiuo, en caso que
la muestia sea liquiua, o soliuo-liquiuo, si la muestia está en estauo soliuo.
La eficacia ue la extiaccion uepenue ue las conuiciones expeiimentales. Poi
ejemplo, el conteniuo fenolico en almenuias es ties veces supeiioi cuanuo
la extiaccion se iealiza a Suº C que cuanuo se utilizan 2SºC. Los
extiactantes más utilizauos son metanol o etanol aciuificauos. Aunque el
piimeio es más eficaz, en la inuustiia alimentaiia suele utilizaise más el
etanol paia evitai la toxiciuau uel metanol. Auemás ue estos extiactantes,
se han utilizauos otios como acetato ue etilo, uietil etei, acetona, n-hexano
e isooctano, entie otios. Noimalmente se usa una extiaccion secuencial con
una piimeia etapa agua-uisolvente oigánico paia sepaiai, poi ejemplo, los
áciuos fenolicos solubles. Los áciuos fenolicos que están enlazauos a las
células meuiante enlaces ésteies o glucosiuicos no se extiaen uiiectamente
en uisolventes oigánicos, sienuo necesaiio libeiailos pieviamente
meuiante hiuiolisis básica y¡o áciua. En geneial se utiliza hiuiolisis básica
con hiuioxiuo souico entie 2 y 1u N, con un tiempo ue incubacion ue hasta
16 h y, a veces, bajo coiiiente ue nitiogeno |18j. Bespués ue la hiuiolisis
básica pueue utilizaise hiuiosis áciua paia completai la libeiacion ue los
compuestos fenolicos. También se ha uesciito la libeiacion ue compuestos
fenolicos meuiante enzimas. La extiaccion con fluiuos supeiciiticos,
utilizanuo uioxiuo ue caibono, es una alteinativa útil paia evitai el uso ue
uisolventes oigánicos |19j aunque es necesaiio utilizai un mouificauoi
como metanol paia aumentai la polaiiuau ue meuio. También se han
uesciito la extiaccion con ultiasoniuos |2uj y la extiaccion asistiua con
enzimas |21j. Se ha piopuesto un autoanalizauoi paia el fiaccionamiento y
cuantificacion en continuo ue los polifenoles piesentes en vinos meuiante
extiaccion en fase soliua y el uetectoi ue uispeision ue luz evapoiativo
|22j.

La ciomatogiafia ue liquiuos es la técnica más utilizaua paia la
sepaiacion ue compuestos fenolicos |4,2S,24j. Noimalmente se usa una
columna C
18
en fase inveitiua con un sistema binaiio ue uisolventes que
consiste en agua aciuificaua y un uisolvente oigánico polai. Es fiecuente
que sea necesaiia la pieconcentiacion y puiificacion pievia, especialmente
Intiouuccion


19
en el caso ue muestias con matiices complejas. Esta etapa pueue iealizaise
utilizanuo paiticion liquiuo-liquiuo con un uisolvente inmiscible o
extiaccion en fase soliua. Paia la utilizacion ue esta técnica pueuen usaise
soibentes como C
18
, copolimeios ue stiieno-uivinilbenceno o uistintos tipos
ue iesinas |2S,26j. La fotometiia es la técnica que más se ha empleauo
como sistema ue ueteccion en estas sepaiaciones ciomatogiáficas, aunque
la espectiometiia ue masas (NS) es la más útil con este fin |27j.

La ciomatogiafia ue gases (uC) también se ha utilizauo paia la
sepaiacion e iuentificacion ue compuestos fenolicos, sienuo necesaiia la
ueiivatizacion meuiante metilacion, tiifluoiacetilacion o conveision a
ueiivauos tiimetilsililo paia foimai compuestos volátiles, y la utilizacion ue
NS como sistema ue ueteccion |28,29j. No obstante, la piepaiacion ue las
muestias en uC es bastante pioblemática ya que hay que eliminai los
lipiuos uel extiacto, libeiai los fenolicos ue sus enlaces ésteies y
glicosiuicos y pioceuei a ueiivatizacion.

La electiofoiesis capilai es otia técnica alteinativa paia la
sepaiacion y ueteiminacion ue compuestos fenolicos uebiuo a su eficacia
en la sepaiacion, iapiuez uel pioceso y bajo consumo ue muestia y
ieactivos, aunque una limitacion fiente a las técnicas ciomatogiáficas es los
ielativamente altos limites ue ueteccion obteniuos |Suj. Se han uesciito
algunos métouos paia la ueteiminacion ue antocianinas y flavonoiues
|S1,S2j.

Existe una vaiieuau ue métouos fotométiicos que peimiten obtenei
una estimacion apioximaua uel conteniuo total ue polifenoles en una
muestia, peio no uan infoimacion cuantitativa ue los componentes
inuiviuuales. El métouo más iepiesentativo ue este giupo es el ensayo
Folin-Ciocalteu |SSj que utiliza áciuo gálico como polifenol estánuai. Como
se uiscute en el Capitulo S ue esta Nemoiia, la piincipal limitacion ue estos
métouos es su escasa selectiviuau, uanuo valoies elevauos ue polifenoles
totales ya que otias especies ieuuctoias piesentes en la muestia
contiibuyen a la señal analitica.

Finalmente, cabe inuicai que se ha uesciito el uso ue uiveisas
técnicas espectioscopicas, tales como NNR, NS y NIR paia la eluciuacion ue
estiuctuias y la caiacteiizacion ue compuestos fenolicos aislauos ue la

2u
matiiz ue la muestia, peio sin sepaiacion pievia ue los componentes
inuiviuuales |4j.


2. Antioxidantes sintéticos

Como se inuico anteiioimente, los antioxiuantes sintéticos son un
giupo ue compuestos fenolicos cuyos componentes más iepiesentativos
son BBA, BBT y uP. Aunque estos compuestos han siuo ampliamente
utilizauos como auitivos alimentaiios, su uso está muy limitauo en la
actualiuau uebiuo a sus potenciales piopieuaues toxicas. Poi ejemplo, el
estuuio ue la citotoxiciuau uel BBA en tejiuos ue animales |S4j ha
uemostiauo que este compuesto inuuce la apoptosis ue las células
meuiante la libeiacion uiiecta ue citociomo C.

Se han uesciito numeiosos métouos paia la sepaiacion
iuentificacion y cuantificacion ue estos compuestos en alimentos |SSj. La
sepaiacion ue la matiiz ue la muestia se ha llevauo a cabo piincipalmente
meuiante extiaccion con uisolventes, sienuo el metanol uno ue los más
utilizauos. La ciomatogiafia ue liquiuos es la técnica más utilizaua paia la
sepaiacion entie los uistintos antioxiuantes, usanuo geneialmente la
espectioscopia 0v como sistema ue ueteccion y, en menoi piopoicion, la
NS |SS,S6j. También se han piopuesto algunos métouos meuiante uC con
el uetectoi ue ionizacion ue llama o con NS paia la cuantificacion |SSj.


FLUORIMETRÍA DE LARGA LONGITUD DE ONDA

El uso ue fluoiofoios ue laiga longituu ue onua (LWFs) pueue
constituii una alteinativa útil paia mejoiai la selectiviuau ue las
ueteiminaciones analiticas fiente a los fluoiofoios convencionales que
piesentan sus caiacteiisticas luminiscentes en la zona 0v-visible. La
piincipal piopieuau ue los LWFs es que su emision se piouuce a longituues
ue onua supeiioies a 6uu nm, uonue piácticamente no existen piocesos ue
absoicion o emision ue compuestos piesentes en la matiiz ue la muestia.
0tias ventajas que ofiecen estos fluoiofoios es que se ieuucen
consiueiablemente los pioblemas asociauos con la posible uegiauacion ue
la muestia, con inteifeiencias uebiuas a señales Raman y con piocesos ue
Intiouuccion


21
inhibicion no iauiantes ya que, noimalmente, la fluoiescencia ue los LWFs
tiene una viua meuia muy coita |S7j. La utiliuau ue los LWFs se ha
uemostiauo en numeiosas aplicaciones, piincipalmente en el análisis
biologico, en el que la fluoiescencia ue la matiiz ue la muestia en la iegion
visible uel espectio es fiecuentemente una fuente ue inteifeiencias cuanuo
se utilizan fluoiofoios convencionales.

Paia que un compuesto emita fluoiescencia a laiga longituu ue
onua, el piimei iequisito es que su estiuctuia sea iigiua, con numeiosos
enlaces conjugauos o anillos aiomáticos fusionauos. El inconveniente que
piesenta un sistema con una elevaua conjugacion es que pueue alteiaise
fácilmente cuanuo se somete a excitacion luminosa. 0tias limitaciones que
fiecuentemente piesentan estos compuestos son su pequeño
uesplazamiento Stokes, su escasa solubiliuau incluso en uisolventes
oigánicos y su faciliuau paia oxiuaise. No obstante, a pesai ue estos
inconvenientes, existe un gian inteiés poi el uso analitico ue estos
compuestos, habiénuose sintetizauo nuevos LWFs que evitan, al menos
paicialmente, estas limitaciones.

El giupo que pueue consiueiaise más iepiesentativo ue LWFs lo
foiman los fluoiofoios tipo cianina cuya estiuctuia básica consiste en uos
anillos aiomáticos o heteiociclicos uniuos poi una cauena polimetina con
uobles enlaces conjugauos, como muestia la Figura 4. Las banuas ue
excitacion ue estos fluoiofoios se encuentian en la zona ue 6uu-9uu nm,
peio pueuen conseguiise uesplazamientos a mayoies longituues ue onua
intiouucienuo un giupo vinilo (-CB=CB-) en la cauena polimetina. Algunos
inconvenientes ue estos compuestos son su escasa ieactiviuau paia
enlazaise a los analitos, su bajo ienuimiento cuántico y su faciliuau paia
foimai agiegauos en meuio acuoso, lo que causa una iápiua uisminucion ue
su fluoiescencia |S8j. No obstante, el compoitamiento ue estos compuestos
como ieactivos analiticos pueue mejoiaise en piesencia ue suifactantes o
ue uisolventes oigánicos. También se ha utilizauo la mouificacion ue su
estiuctuia moleculai, intiouucienuo giupos alquilsulfonato, paia mejoiai
su solubiliuau en agua, su ienuimiento cuántico y su estabiliuau
fotoquimica, o bien, la intiouuccion ue giupos ieactivos, tales como
isotiocianatos, lo que peimite la foimacion ue enlaces covalentes.



22






























Figura 4. Estiuctuia ue los fluoiofoios tipo cianina. (1) Estiuctuia básica,
(2) CyS, (S) veiue ue inuocianina.


Bentio ue los fluoiofoios cianina utilizauos con fines analiticos cabe
citai el CyS y el veiue ue inuocianina (ICu), también uenominauo IR-12S,
cuyas estiuctuias se muestian en la Figura 4. El CyS ha siuo ampliamente
utilizauo en CE |S9-42j y en el uesaiiollo ue sensoies |4S-Suj, mientias que
el ICu fue inicialmente utilizauo en el campo ue la imagen clinica uebiuo a
su escasa toxiciuau |S1j. Aunque este compuesto es muy poco fluoiescente
en meuio acuoso, su fluoiescencia aumenta al enlazaise a algunos
compuestos, tales como pioteinas |S8,S1j. En esta memoiia se ha utilizauo
el ICu paia uesaiiollai un métouo paia la ueteiminacion ue polifenoles
totales.
Intiouuccion


2S
0tio giupo ue LWFs lo constituye los fluoiofoios tipo oxacinas, cuya
estabiliuau fotoquimica es mejoi que la ue los fluoiofoios cianina. En la
Figura 5 se muestian las estiuctuias ue algunas oxacinas iepiesentativas,
tales como el violeta ue ciesilo, azul Nilo y 0xacina 7Su, uonue pueue
obseivaise que piesentan estiuctuias más compactas que las ue los
fluoiofoios cianina. La utiliuau analitica ue estos compuestos ha siuo
ampliamente uesciita |S2-62j. El violeta ue ciesilo se ha utilizauo en un
métouo ciomatogiáfico uesciito en esta Nemoiia paia la ueteiminacion ue
flavonoiues. También cabe citai como LWFs utilizauos con fines analiticos a
uos ueiivauos iouamina, la Rouamina 8uu y el Rojo Texas, cuyas
estiuctuias se muestian en las Figura 6, y a la naftofluoiesceina, las
ficobilipioteinas y a los ueiivauos escuaiina, aunque su uso como ieactivos
analiticos ha siuo ielativamente limitauo en los últimos años |6S-66j.


Figura 5. Estiuctuia ue los fluoiofoios tipo oxacina. (1) violeta ue Ciesilo
(R: -B
2
) y Azul Nilo (R:-(C
2
BS)
2
), (2) 0xacina 7Su.














Figura 6. Estiuctuia quimica ue: (1) Rouamina 8uu, (2) RojoTexas.

24
0n giupo especial ue LWFs lo foiman algunos complejos ue
iutenio(II), cuya viua meuia ue la fluoiescencia es mucho mayoi que la ue
los LWFs oigánicos. En geneial emiten en el inteivalo 61u-6Su nm, aunque
sus piopieuaues espectioscopicas y quimicas vaiian según el liganuo. Los
complejos ue iutenio(II) se han utilizauo piincipalmente paia el uesaiiollo
ue nuevos sistemas sensoies. Poi ejemplo, se han uesciito sensoies ue pB
basauos en quelatos con caiacteiisticas luminiscentes sensibles al pB y en
la meuiua ue la intensiuau luminiscente |67-69j o ue la uuiacion ue la
luminiscencia |7u-72j.

Los LWFs han siuo muy utilizauos en piocesos ue tiansfeiencia ue
eneigia (fluoiescence iesonance eneigy tiansfei, FRET) con fines
analiticos. En estos piocesos, noimalmente, un compuesto fluoiescente
actúa como uauoi ue eneigia no iauiante a un compuesto aceptoi, que
pueue sei fluoiescente o no fluoiescente, oiiginánuose la uisminucion ue la
emision fluoiescente ue la molécula uauoia. Bebe existii un consiueiable
solapamiento entie el espectio ue emision uel uauoi y el ue absoicion uel
aceptoi, no sienuo necesaiio que exista una inteiaccion fisica entie ambos
si la uuiacion ue la fluoiescencia uel uauoi es mayoi que la uel pioceso ue
tiansfeiencia ue eneigia. Se han uesciito uiveisas aplicaciones analiticas ue
estos piocesos utilizanuo quelatos ue Ru(II) o ue lantániuos como uauoies
ue eneigia |6S,7S-78j. Como ejemplo cabe citai la piepaiacion ue
nanoesfeias luminiscentes como maicauoies paia multiueteccion usanuo
un quelato ue iutenio(II) (uauoi) uisuelto con vaiios fluoiofoios cianina
(aceptoies) en un polimeio poliaciilonitiilo |7Sj. Las nanoesfeias emiten
uoble luminiscencia, una pioceuente uel uauoi y otia ue aceptoi, vaiianuo
la eficacia ue FRET al vaiiai la concentiacion ue aceptoi, lo que pueue
monitoiizaise miuienuo el máximo ue emision y la caiua ue la
luminiscencia.

El uso ue LWFs como ieactivos ueiivatizantes en ciomatogiafia ue
liquiuos ha siuo bastante iestiingiuo. Se ha piopuesto el azul Nilo paia la
ueiivatizacion piecolumna en la ueteiminacion ue vaiios compuestos
caiboxilicos como los áciuos benzoico, acético, fenilacético y hexanoico
|S7j. La aplicabiliuau piáctica uel sistema se uemostio meuiante la
ueteiminacion ue áciuo fenilacético en plasma |S8j. La Rouamina 8uu se ha
utilizauo como auitivo ue la fase movil paia la cuantificacion ue áciuo
valpioico en plasma utilizanuo ueteccion inuiiecta ue la fluoiescencia |79j.
Intiouuccion


2S
Esta metouologia peimite obtenei una iespuesta univeisal cuanuo el
fluoiofoio foima paite ue la fase movil y piesenta cieita afiniuau poi la fase
estacionaiia ue foima que, al inyectai los analitos en la columna se obtiene
una iespuesta uel uetectoi uebiua a la iuptuia uel equilibiio ue
uistiibucion uel fluoiofoio entie las fases movil y estacionaiia. FRET
también se ha utilizauo en un sistema postcolumna paia ueteiminai biotina
en plasma humano usanuo una ficoeiitiina conjugaua a biotina y CyS uniua
a estieptaviuina |64j.

Los LWFs han encontiauo mayoi aplicacion en CE |S9-41,8u,81j. Se
han uesciito uiveisos LWFs mouificauos paia la ueiivatizacion con giupos
amino ue aminoáciuos y pioteinas. Poi ejemplo, existen uiveisos ueiivauos
comeiciales ue LWFs paia el maicaje ue giupos amino, como el ueiivauo ue
CyS utilizauo paia la ueteiminacion ue plaguiciuas aminoáciuos fosfoiauos
en suelos meuiante ueiivatizacion off-line pievia a la sepaiacion meuiante
CE |82j. Poi el contiaiio, la uisponibiliuau ue LWFs como ieactivos
ueiivatizantes ue giupos caiboxilo es más iestiingiua. Cabe citai un
coloiante cianina polimetina piopuesto paia uniilo a áciuos giasos, con un
giupo amino aiomático que se enlaza covalentemente a los analitos |8Sj.
También existen algunas aplicaciones en las que los LWFs se han utilizauo
como maicauoies no covalentes paia la ueteiminacion ue pioteinas
meuiante CE |84,8Sj. Esta alteinativa es más simple y iápiua que el maicaje
covalente, uonue noimalmente se iequieie un contiol estiicto uel pB.
Auemás, su uso en CE minimiza la limitacion encontiaua en ciomatogiafia
ue liquiuos en la que el exceso ue fluoiofoio pueue queuai ieteniuo en la
zona hiuiofobica ue la columna |86j. Como ejemplo uel uso uel maicaje no
covalente en CE cabe citai un estuuio compaiativo paia pioteinas con
coloiantes escualeno en las moualiuaues pie-columna y en columna,
encontianuo mejoies iesultauos en el segunuo caso |8Sj.

También se ha uesciito el uso ue LWFs en ciomatogiafia
electiocinética micelai (NEKC) meuiante ueteccion ue fluoiescencia
inuiiecta, opcion que es útil cuanuo los analitos no poseen piopieuaues
fluoiescentes auecuauas o un giupo funcional que pueua enlazaise a un
fluoiofoio. El mecanismo ue ueteccion implica la peituibacion uel complejo
fluoiofoio-micela poi el analito, uebienuo sei uifeiente la intensiuau ue la
fluoiescencia que piesenta el fluoiofoio en la fase micelai a la que piesenta
en fase acuosa. Esta técnica se ha utilizauo, poi ejemplo, paia ueteiminai

26
explosivos en suelo meuiante un coloiante cianina |87j usanuo un
miciochip con una longituu ue sepaiacion ue 6S mm.


LUMINISCENCIA SENSIBILIZADA DE LANTÁNIDOS

Los iones lantániuos, especialmente teibio(III) y euiopio(III)
foiman quelatos con liganuos multiuentauos, piincipalmente con giupos
uauoies ue oxigeno caigauos negativamente, que piesentan especiales
piopieuaues luminiscentes en uisolucion uebiuo a un pioceso ue
tiansfeiencia ue eneigia intiamoleculai uesue el estauo tiiplete excitauo
uel liganuo al ion lantániuo. Constituye un pioceso ue luminiscencia
sensibilizaua muy iápiuo que, noimalmente, no está contiolauo poi
uifusion ya que el lantániuo está uniuo al liganuo meuiante enlace
cooiuinauo |88j.

Aunque pouiia consiueiaise que la luminiscencia sensibilizaua ue
lantániuos es un tipo especial ue fosfoiescencia sensibilizaua a tempeiatuia
ambiente, existen uifeiencias básicas entie ambos piocesos uebiuo a que el
piimeio es intiamoleculai y el segunuo es inteimoleculai. En este último,
una especie con una estiuctuia capaz ue piesentai un ciuce entie sistemas
eficaz uesue el estauo singlete excitauo al tiiplete excitauo, pueue tiansfeiii
su eneigia uesue este estauo a un compuesto como biacetilo o 1,4-
uibiomonaftaleno, el cual se excita pasanuo al estauo tiiplete, con la
consiguiente emision ue fosfoiescencia. Bebiuo a que se tiata ue un
pioceso inteimoleculai en uisolucion, el oxigeno pueue inhibii fácilmente
los estauos tiipletes uel uauoi y uel aceptoi poi lo que es esencial la
uesoxigenacion uel meuio. Poi el contiaiio, el caiáctei intiamoleculai ue la
luminiscencia sensibilizaua ue lantániuos hace que el pioceso sea menos
susceptible a la inhibicion colisional uel estauo tiiplete uel uauoi poi el
oxigeno. Algunas ue las iazones que justifican la aplicabiliuau analitica ue
esta técnica son las siguientes:

1) Los iones lantániuos piesentan una buena estabiliuau y su
toxiciuau es baja ya que tienen una escasa absoicion en el
tiacto gastiointestinal e incluso, aunque se inyecten, no pueuen
penetiai en las células.

Intiouuccion


27
2) Pueuen foimai quelatos muy luminiscentes uebiuo a los
piocesos anteiioimente inuicauos ue tiansfeiencia ue eneigia
uesue el liganuo al lantániuo. Estos piocesos uan lugai a que
los quelatos piesenten un gian uesplazamiento Stokes evitanuo
pioblemas ue solapamiento entie las banuas ue excitacion y
emision, tales como los que piesentan fluoiofoios
convencionales como la fluoiesceina y que obliga a ieuucii el
ancho ue banua ue las ienuijas, con la consecuente ieuuccion
ue la señal fluoiescente.

3) La emision uel lantániuo piesenta una gian selectiviuau
espectial, con banuas ue emision muy estiechas, uebiuo a que
este pioceso se piouuce a tiavés ue los niveles iesonantes uel
ion lantániuo.

4) También piesentan muy buena selectiviuau tempoial uebiuo a
la ielativamente laiga uuiacion ue la emision ue algunos
quelatos lantániuos, lo que peimite iealizai la meuiua analitica
meuiante el mouo tiempo iesuelto, cuanuo la emision
fluoiescente ue inteifeientes fluoiescentes se ha anulauo. Esta
caiacteiistica también mejoia la sensibiliuau ya que la meuiua
se pueue iealizai meuiante la integiacion ue la señal uuiante
un tiempo ueteiminauo.

5) La emision se piouuce a longituues ue onua ielativamente
laigas, a uifeiencia ue las banuas que piesentan la
fluoiescencia ue fonuo ue la muestia, las cuales apaiecen ue
menoies longituues ue onua.

Paia que se piouuzca una tiansfeiencia ue eneigia eficaz uesue el
liganuo-analito al ion lantániuo ueben cumpliise los siguientes iequisitos:
1) las tiansiciones no iauiantes uel liganuo (S
1
S
u
o T
1
T
u
) ueben se
minimas; 2) la eneigia uel nivel ue iesonancia uel ion lantániuo uebe sei
ligeiamente infeiioi a la uel estauo tiiplete excitauo uel liganuo, ue foima
que exista una elevaua piobabiliuau ue que se piouuzca la tiansfeiencia ue
eneigia; y S) no ueben piouuciise tiansiciones no iauiantes uel ion
lantániuo excitauo.


28
Existen uiveisos factoies, tales como el uso ue agentes sineigéticos,
tensoactivos, pB, co-luminiscencia y átomos pesauos, que contiibuyen a
que se cumplan estos iequisitos |89j. Poi ejemplo, uebiuo al elevauo
númeio ue cooiuinacion uel teibio(III), al foimai quelatos con cieitos
liganuos pueuen queuai sitios ue cooiuinacion libies que pueuen sei
ocupauos poi moléculas ue agua, lo que favoiece la inhibicion ue la
luminiscencia uebiuo a piocesos ue uesactivacion no iauiantes. Este efecto
negativo se evita utilizanuo un segunuo liganuo que uesplaza a las
moléculas ue agua ue la esfeia ue cooiuinacion uel ion lantániuo,
ejeicienuo un efecto sineigético en la luminiscencia sensibilizaua. Con este
fin se utilizan aminopolicaiboxilatos y el oxiuo ue tii-n-octil fosfina (T0P0)
ya que tienen fueites piopieuaues cooiuinantes y pueuen llenai los huecos
vacios ue la esfeia ue cooiuinacion uel lantániuo. La piesencia ue ties
cauenas octilo uauoias en la molécula ue T0P0 piotege tanto al teibio(III)
como al liganuo ue posibles colisiones que oiiginen uesactivaciones no
iauiantes. La piesencia ue tensoactivos en el sistema también piotege al
quelato ue piocesos ue inhibicion.

0tio pioceso que pueue mejoiai la intensiuau ue la luminiscencia
sensibilizaua es el fenomeno ue la co-luminiscencia uebiua a un aumento
ue la excitacion uel ion lantániuo favoieciua poi la absoicion ue quelatos
que no emiten luminiscencia peio que tiansfieien la eneigia absoibiua al
quelato uel lantániuo. Este pioceso se ha utilizauo en esta Nemoiia paia la
ueiivatizacion postcolumna ue flavonoiues meuiante la foimacion ue
quelatos ue aluminio y teibio.

Las especiales caiacteiisticas ue la luminiscencia sensibilizaua ue
teibio han uauo lugai a un elevauo númeio ue métouos paia la
ueteiminacion ue compuestos oigánicos basauos en la foimacion ue
quelatos estables con analitos-liganuos que contienen átomos ue oxigeno
caigauos negativamente |88j. También se ha uesciito su utiliuau en
bioensayos, tales como el inmunoensayo |9uj, existienuo ensayos
comeiciales como el sistema BELFIA |91j.

0tio campo ue aplicacion analitica ue la luminiscencia sensibilizaua
ue teibio ha siuo su uso como sistema ue ueteccion en ciomatogiafia ue
liquiuos meuiante ieacciones ue ueiivatizacion pie- o postcolumna. En la
Tabla 3 se muestian algunas ue las aplicaciones uesciitas |92-99j, uonue
Intiouuccion


29
pueue obseivaise que se consiguen limites ue ueteccion muy bajos en el
análisis ue muestias con matiices bastante complejas. En esta Nemoiia se
uesciiben uos métouos en los que se utiliza el teibio(III) como
ueiivatizante postcolumna con iesultauos muy satisfactoiios como se
uiscute posteiioimente.

Tabla3. Aplicaciones ue los iones ue teibio como ieactivos ueiivatizantes
en ciomatogiafia ue liquiuos.

Analito
Fase
Novil
L0B Nuestia Refeiencia
0chiatoxin A RP S µN Queso |92j
0iotato RP 1 nN 0iina |9Sj
ulutation RP 2u nN 0iina |94j
L-cisteina RP u.1S µN 0iina |9Sj
Compuestos
fenolicos
RP u.u16 - u.S µg mL
-1
vino |96j
Cipiofloxacina RP u.1 µN Biologica |97j
Flumequina RP
1.1 ng mL
-1
1S ng mL
-1
Pollo |98j
Fluoioquinolonas RP 2 - 9S ng mL
-1
Leche |99j



DISPERSIÓN DE LA RADIACIÓN

La uispeision ue la iauiacion es un fenomeno optico que ha siuo
ampliamente utilizauo como sistema ue ueteccion en los métouos
analiticos, aunque iecientemente ha aumentauo el inteiés poi su uso uesue
la intiouuccion ue nanomateiiales en uichos métouos.

Según el tamaño ue las paiticulas (u) que inteiaccionan con los
fotones ue la iauiacion y ue la longituu ue onua (i) ue la iauiacion
inciuente, pueuen uistinguiise uifeientes fenomenos ue uispeision. La
uispeision Rayleigh (u < u.S i) la oiiginan paiticulas cuyo uiámetio meuio
es menoi que la longituu ue onua ue la iauiacion, piouuciénuose una
uispeision piácticamente simétiica en touas las uiiecciones. Al aumentai el
tamaño ue la paiticula, la iauiacion uispeisaua se uesplaza en el sentiuo ue
la iauiacion tiansmitiua, uanuo lugai a las uispeisiones Tynuall (u ~ i) y
Nie (u >> i). Existen otios fenomenos en los que, auemás ue la uispeision,

Su
se piouuce un cambio en la fiecuencia ue la iauiacion, como ocuiie en el
efecto Raman.

Las uos técnicas clásicas basauas en el fenomeno ue la uispeision
sin cambio ue fiecuencia son la tuibiuimetiia y la nefelometiia, cuya
piincipal uifeiencia se encuentia en la foima ue iealizai las meuiuas ue
uispeision. En tuibiuimetiia se obtiene la ielacion entie la intensiuau ue la
iauiacion tiansmitiua y la intensiuau ue la iauiacion inciuente, mientias
que en nefelometiia se miue la iauiacion uispeisaua que foima un ángulo
(< 9uº) con iespecto a la iauiacion inciuente. En geneial, los limites ue
ueteccion que se consiguen con nefelometiia son más bajos. Estas técnicas
son ampliamente utilizauas en el laboiatoiio clinico como sistemas ue
ueteccion en métouos inmunoquimicos paia la ueteiminacion ue pioteinas,
tales como feiiitina |1uuj y fibiina soluble |1u1j. El fenomeno ue
uispeision ue la iauiacion también se ha utilizauo como sistema ue
ueteccion en uiveisas ueteiminaciones ciomatogiáficas |1u2-1uSj.

La técnica ue uispeision ue iauiacion iesonante (RLS) utiliza un
espectiofluoiimetio convencional en el que se iealiza el baiiiuo simultáneo
ue los monociomauoies ue excitacion y emision con un Ai = u |1u6j,
obtenienuo una señal intensa en las pioximiuaues ue la zona ue máxima
absoicion ue una especie que piesente piopieuaues absoibentes. La
iauiacion uispeisaua en estas conuiciones pueue consiueiaise que sigue la
teoiia Rayleigh en la que se consiueia que touos los electiones en una
paiticula oscilan con la misma fase y fiecuencia que la onua
electiomagnética inciuente y su oscilacion colectiva ua lugai a un gian
momento uipolai eléctiico oscilante que oiigina iauiacion uispeisaua. No
obstante las señales RLS obteniuas con un espectiofluoiimetio pueuen
incluii, auemás ue la uispeision Rayleigh, otios tipos ue uispeision ue
iauiacion como las uispeisiones Nie y Tynuall.

Esta técnica es muy simple y se ha aplicauo a la ueteiminacion ue
sustancias que foiman agiegauos moleculaies con un ciomofoio, poi
ejemplo, hepaiina con azul ue metileno |1u7j, albúmina con iojo ue
piiogalol |1u6j y áciuos nucleicos con moiina |1u9j. Actualmente tiene un
gian inteiés paia la monitoiizacion ue fenomenos ue agiegacion utilizanuo
nanopaiticulas ue oio o ue plata, cuyo tamaño es menoi ue 2u veces la
longituu ue onua ue la iauiacion inciuente. Estas nanopaiticulas piesentan
Intiouuccion


S1
absoicion en la iegion visible, piouuciénuose un uesplazamiento
batociomico al aumentai su tamaño uebiuo a la agiegacion. La utiliuau ue
este fenomeno se ha puesto ue manifiesto en inmunoensayo y en estuuios
ue hibiiuacion ue áciuos nucleicos |11uj. En esta Nemoiia se utiliza la RLS
como sistema ue ueteccion paia la ueteiminacion ue antioxiuantes
meuiante la foimacion ue nanopaiticulas ue oio.


SISTEMAS DE FLUJO

En las investigaciones que se piesentan en esta Nemoiia se utilizan
sistemas ue análisis en flujo paia las ueteiminaciones basauas en
ueiivatizacion postcolumna en ciomatogiafia ue liquiuos y un sistema ue
mezcla ue flujo ueteniuo en las ueteiminaciones sin sepaiacion
ciomatogiáfica. Poi ello, aunque bievemente, se comentan las piincipales
caiacteiisticas ue ambos sistemas.

La técnica ue análisis en flujo, conociua como FIA (flow injection
analysis), foima paite ue las técnicas automáticas ue análisis con flujo no
segmentauo, en las que las ueteiminaciones se iealizan ue foima continua
sin utilizai buibujas ue aiie paia la sepaiacion entie las uistintas zonas
tianspoitauas a lo laigo uel sistema. Noimalmente se utilizan muestias
liquiuas que se inseitan uiiectamente en el sistema uinámico (Figura 7 A)
a tiavés uel cual son tianspoitauas, puuienuo incluii ieacciones quimicas o
bioquimicas y sistemas ue sepaiacion y¡o pieconcentiacion. Se utiliza un
uetectoi continuo, piovisto geneialmente ue una célula ue flujo, paia la
obtencion ue las señales tiansitoiias caiacteiisticas ue la técnica (Figura 7
B). Los métouos uesaiiollauos meuiante FIA en esta veision simple son
métouos cinéticos ue tiempo fijo y, poi tanto, ni el equilibiio fisico
(homogenizacion ue la poicion uel fluiuo) ni el quimico (equilibiio ue la
ieaccion) se han alcanzauo en el momento ue la ueteccion, poi lo que el
tiempo ue opeiacion ha ue sei muy iepiouucible.

Las caiacteiisticas más uestacables ue la técnica son:

• veisatiliuau: Entie otios usos cabe uestacai su utilizacion como
simple inteifaz entie muestia e instiumento, con inteicalacion
tanto ue etapas quimicas o bioquimicas como ue sepaiacion y¡o

S2
pieconcentiacion o ue ambas, la implantacion ue sensoies
quimicos y¡o bioquimicos en flujo, su acoplamiento con
ciomatogiafos, o con uetectoies ICP-AES, ICP-NS, FTIR, NS y, su
uso paia el seguimiento ue piocesos inuustiiales.





















Figura 7. 0niuaues básicas ue un sistema FI (A) y señales tiansitoiias
tipicas ue la técnica (B).


• Rapiuez: Es una consecuencia ue la ausencia ue contaminacion
entie muestias inyectauas. Las caiacteiisticas geométiicas ue
los sistemas favoiecen la uispeision iauial en uetiimento ue la
axial, y como iesultauo se obtiene la eliminacion ue la fase ue
lavauo entie muestias sucesivamente inyectauas. Se obtienen
fiecuencias ue muestieo en conuiciones noimales ue 6u - 1uu
h
-1
.
• Piecision: Estuuios sobie este paiámetio analitico han
peimitiuo establecei como más común, un valoi ue la
uesviacion estánuai ielativa ue los métouos FIA infeiioi al 2%
|111j
Intiouuccion


SS
• Coste: Bajo, ya que con estos sistemas se suele tiabajai a bajas
piesiones lo que peimite piescinuii ue uniuaues (bombas,
válvulas, etc) ue alta piesion ue un piecio mucho mayoi.

La técnica ue mezcla ue flujo ueteniuo |112,11Sj peimite ue una
foima muy simple la automatizacion ue las meuiuas cinéticas. Como se
muestia en el esquema ue la Figura 8, los ieactantes ue un sistema quimico
se uistiibuyen entie uos jeiingas, uenominauas jeiingas ue impulsion, y son
conuuciuos a gian velociuau hacia la cámaia ue mezcla que, noimalmente,
es también la celua ue obseivacion uel instiumento. El flujo se uetiene
biuscamente meuiante una teiceia jeiinga, uenominaua jeiinga ue paiaua,
peimitienuo la obtencion iápiua ue la cuiva cinética señal-tiempo, cuanuo
el sistema ha alcanzauo el estauo estacionaiio. Neuiante un sopoite
infoimático auecuauo se obtienen uatos cinéticos, tales como la velociuau
inicial ue la ieaccion, amplituu ue señal y, en su caso, peiiouo ue inuuccion.











Figura 8. Esquema ue la técnica ue flujo ueteniuo

Algunas caiacteiisticas ue esta técnica se iesumen a continuacion:
1) mezcla iápiua, completa y automática ue los ieactantes, 2) bajo consumo
ue los mismos, S) aplicable a sistemas iápiuos y lentos, 4) gian piecision, S)
minimizacion ue inteifeiencias, 6) obtencion iápiua ue iesultauos, y 7)
automatizacion ue la etapa ue meuiua. Estas caiacteiisticas peimiten
obtenei una gian velociuau ue muestieo, lo que justifica su utilizacion en
análisis ue iutina. En algunos sistemas, la etapa ue meuiua iequieie solo
unos segunuos e, incluso, uécimas ue segunuo. En sus inicios, la técnica ue
mezcla ue flujo ueteniuo se oiiento piincipalmente hacia el estuuio ue la
cinética y mecanismos ue ieacciones iápiuas. No obstante, su utiliuau en

S4
análisis ue iutina ha siuo suficientemente uemostiaua |114-116j, sienuo su
campo ue aplicacion extiaoiuinaiiamente amplio.


QUIMIOMETRÍA

El teimino quimiometiia fue utilizauo poi piimeia vez en 1972 poi
el cientifico sueco Wolu, cuanuo hizo uso ue uifeientes pioceuimientos
estauisticos paia el tiatamiento ue uatos quimicos. En esa uécaua, junto con
el quimico analitico Kowalski y otios investigauoies ameiicanos cieaion la
Inteinational Chemical Society (ICS) en cuyo ámbito, canalizaion el iápiuo
y amplio uesaiiollo ue la quimiometiia como heiiamienta ue utiliuau en el
piocesauo ue uatos quimicos. Según la ICS, la quimiometiia es la uisciplina
cientifica que peimite aplicai heiiamientas estauisticas y matemáticas paia
uiseñai y¡o seleccionai expeiimentos y pioceuimientos ue meuiua, asi
como paia extiaei la máxima cantiuau ue infoimacion quimica a paitii ue
los uatos uisponibles |117-12uj. Su utiliuau se ve especialmente ieflejaua
uentio ue la Quimica Analitica, uonue la iuentificacion, clasificacion e
inteipietacion ue los uatos piopoicionauos poi las técnicas más actuales ue
instiumentacion analitica pueuen llegai a sei una taiea especialmente
abiumauoia.

La instiumentacion analitica actual piopoiciona una ingente
cantiuau ue infoimacion, geneialmente agiupaua en foima ue matiices
multiuimensionales ue uatos que a su vez se pouiá coiielacionai con la
concentiacion ue los componentes ue una muestia, uenominaua objeto.
Caua muestia pueue coiielacionaise con un sinfin ue meuiuas, uesignauas
como vaiiables. La implantacion uel análisis multivaiiante como técnica ue
tiatamiento ue uatos conlleva a una mejoia sustancial en la caliuau ue la
infoimacion analitica ue la muestia en estuuio, o sobie la similituu o no
entie muestias en estuuios ue clasificacion. Fiecuentemente los uatos
multivaiiantes pueuen sei tiatauos con fines ue uisciiminacion y¡o ue
clasificacion entie muestias. Caua objeto se caiacteiiza poi un conjunto ue
meuiuas que a su vez pueuen iepiesentaise como un vectoi en un espacio
multiuimensional, poi lo que el análisis multivaiiante pueue ayuuai a
visualizai ue foima efectiva uifeientes objetos. La quimiometiia incluye
también metouologias paia el análisis exploiatoiio ue uatos, el uiseño
expeiimental y el mouelauo ue la infoimacion. Nuchas ue las aplicaciones
Intiouuccion


SS
uesaiiollauas implican metouologias multivaiiantes paia la clasificacion ue
poblaciones.

Besue comienzos ue los años 8u el númeio ue aiticulos y ievisiones
sobie quimiometiia ha siuo muy extenso y está ampliamente uistiibuiuo
en el ámbito ue las publicaciones cientificas. Se han uesciito métouos
quimiométiicos que inciuen en las uistintas etapas uel pioceso analitico,
incluyenuo muestieo, optimizacion ue vaiiables, filtiauo ue señales,
ensayos ue simulacion, calibiacion multivaiiante y análisis clasificatoiio. A
continuacion se haiá una uesciipcion muy genéiica ue algunas ue las
técnicas quimiometiicas utilizauas en el uesaiiollo ue esta Tesis Boctoial
en lo iefeiente al pie-tiatamiento ue uatos y al empleo ue metouologias
paia el análisis clasificatoiio.

0na piáctica habitual en el ámbito ue la quimiometiia es el pie-
tiatamiento ue los uatos como paso funuamental pievio a la utilizacion
posteiioi ue otias técnicas, ya sean ue iegiesion o ue clasificacion. En
análisis multivaiiante caua muestia es consiueiaua como un vectoi o como
una matiiz ue uatos en la que en muchas ocasiones pueuen piouuciise
efectos no ueseauos que uistoisionan la secuencia auecuaua ue la
infoimacion suministiaua |121j. 0tio ejemplo significativo ue uistoision
que iequieie etapas ue pie-tiatamiento lo constituye la piesencia ue
huecos en los vectoies o matiices ue uatos, como poi ejemplo en el
tiatamiento ue iegistios ciomatogiáficos. 0sualmente, la piesencia ue
estos huecos en la secuencia ue uatos se coiiesponue con ausencias
significativas en una obseivacion concieta o en la obtencion ue valoies ue
concentiaciones ue analitos poi uebajo ue los limites ue ueteccion ue la
metouologia aplicaua. La utilizacion ue vectoies o matiices ue uatos con
uistoisiones ajenas al objeto ue estuuio conlleva geneialmente a eiioneas
aplicaciones ue las técnicas quimiométiicas y poi tanto a conclusiones no
ueseauas. En estas situaciones, el pie-tiatamiento ue los uatos minimiza
estas uistoisiones y posibilitan la constiuccion ue mouelos quimiométiicos
más simples y iobustos.

Las técnicas ue pie-piocesauo se pueuen uiviuii en uos gianues
giupos: aquellas que usan uiiectamente unos valoies ue iefeiencia
uisponibles paia el pie-piocesauo ue uatos y aquellas que no los usan |122-
128j. Estas últimas se pueuen uiviuii a su vez en otios uos sub-giupos:

S6
métouos ue coiieccion ue la uispeision y métouos ue ueiivatizacion. Los
métouos ue pie-piocesauo uepenuiente ue iefeiencia compienuen una
seiie ue metouologias basauas en ajustes oitogonales ue los uatos iespecto
a la iefeiencia seleccionaua.

Bentio ue las técnicas más comunes paia la coiieccion ue la señal
cabe uestacai las siguientes:

1) El centiauo sobie la meuia, que consiste en cambiai el oiigen ue
la nueva escala ue vaiiables poi la meuia ue la vaiiable antes uel
centiauo. Este tiatamiento no mouifica la vaiianza ue los uatos
expeiimentales.
2) El autoescalauo, que es un centiauo seguiuo ue una
noimalizacion y que se utiliza cuanuo la magnituu ue las señales
o la ielacion señal¡iuiuo vaiia consiueiablemente ue una
vaiiable a otia, sienuo especialmente impoitante en la
exploiacion ue uatos multivaiiante.
S) La coiieccion ue la linea ue base, que se utiliza cuanuo existen
vaiiaciones significativas en la linea ue base entie seiies ue
uatos como poi ejemplo en seiies espectiales. La aplicacion más
simple supone el uso ue algoiitmos ue substiaccion ue señales
con iespecto a ueteiminauas zonas uonue la intensiuau ue la
señal es minima. Se obtienen nuevas matiices con uatos
coiiegiuos que piopician la anulacion ue los efectos ue
uistoision.
4) El suavizauo y filtiauo ue señales, que intenta ieuucii el iuiuo
aleatoiio existente en la señal instiumental meuiante ajuste poi
uesplazamiento ue la meuia ue un pequeño inteivalo ue valoies.
La metouologia ue suavizauo más usaua es la ue Savitzky-uolay,
en la que se usa un mouelo ue ajuste ue minimos cuauiauos con
una funcion polinomica aplicaua sobie una ventana movil ue
uatos.
S) Las técnicas ue ueiivacion ue señales se funuamentan en que la
uifeienciacion matemática peimite acentuai las uifeiencias
conteniuas en la matiiz ue uatos. Poi ello, el cálculo ue la
piimeia y la segunua ueiivaua es un pioceuimiento útil paia
eliminai iuiuos ue fonuo. Las metouologias ue ueiivacion ue
señal más usauas se ueben a Savitzky-uolay y a Noiiis-Williams,
Intiouuccion


S7
que peimiten un suavizauo ue las señales en base a la ueiivacion
ue las señales a tiavés ue la aplicacion ue apioximaciones con
algoiitmos polinomicos. En ueteiminauas ciicunstancias, la
técnica ue noimalizacion ue los uatos expeiimentales pievia al
tiatamiento posteiioi meuiante análisis multivaiiante es una
técnica útil paia el compensauo ue fluctuaciones exteinas al
pioceso ue meuiua.
6) Las técnicas ue coiieccion multiplicativa ue señal (NSC) y ue
vaiiacion estánuai noimalizaua (SNv) son metouologias ue pie-
tiatamiento en las que se suavizan los efectos ue uispeision ue la
iauiacion y ue uifeiencias uebiuas al tamaño ue las paiticulas en
suspension, especialmente útiles cuanuo se piocesan espectios
ue ieflectancia IR en suspensiones.
7) El objetivo ue la metouologia uenominaua coiieccion oitogonal
ue la señal (0SC) es coiiegii la matiiz ue uatos oiiginales
eliminanuo infoimacion oitogonal en la constiuccion ue una
matiiz ue iespuesta.

Como se ha inuicauo anteiioimente, las técnicas analiticas
moueinas geneian gian cantiuau ue infoimacion cualitativa y cuantitativa,
poi lo que con fiecuencia es necesaiio aplicai métouos foimales capaces ue
iesaltai similituues y uifeiencias entie seiies ue objetos. Las técnicas ue
ieconocimiento ue pautas facilitan la iesolucion ue aspectos tales como la
iuentificacion ue las ielaciones entie objetos quimicamente caiacteiizauos
|129-1S6j.

Los objetivos funuamentales uel análisis ue ieconocimiento ue
pautas se centian en la iuentificacion ue ielaciones y¡o vinculos entie
objetos agiupauos o clasificauos según la similituu ue sus uatos
expeiimentales. Bentio ue las técnicas ue ieconocimiento ue pautas existen
uifeientes mouelos ue tiabajo, que se pueue uiviuii en uos gianues giupos:
1) el análisis exploiatoiio y 2) los métouos ue clasificacion, que a su vez se
clasifican en uos mouelos: (a) no supeivisauos, que se utilizan paia uecii si
un conjunto ue pautas (vaiiables) se pueuen uiviuii en giupos, y (b)
supeivisauos, en los que se conocen las clases en que pueue uiviuiise una
muestia, y el objetivo es la clasificacion ue una muestia ue clase
uesconociua poi sus pautas. Como ejemplo ue métouos no supeivisauos
cabe uestacai el análisis ue giupos o "clustei" (CA), mientias que en el

S8
segunuo giupo se encuentian el análisis uisciiminante lineal (LBA) y
cuauiático (QBA), métouo ue los K vecinos más pioximos (KNN), ue
mouelauo suave ue clases análogas inuepenuientes (SINCA) y las ieues
neuionales aitificiales (ANN).

Las técnicas ue análisis exploiatoiio ue uatos se utilizan paia ponei
ue manifiesto y iesaltai infoimacion conteniua en una matiiz ue uatos
multiuimensional. La obseivacion uiiecta ue los uatos expeiimentales, asi
como la utilizacion ue heiiamientas estauisticas simples son insuficientes
paia uesciibii en toua su magnituu las estiuctuias piesentes. Las técnicas
ue exploiacion ue uatos consisten funuamentalmente en uos tipos ue
técnicas: análisis ue componentes piincipales (PCA) y análisis factoiial
(FA), sienuo esta última la utilizaua en el uesaiiollo ue esta Nemoiia.
Ambas técnicas se iequieien paia uefinii estiuctuias que ocupan más ue
ties uimensiones, paia iuentificai tenuencias ocultas piesentes en los uatos
expeiimentales y estuuiai las fuentes o causas ue vaiianza a que obeuecen,
paia obtenei vaiiables latentes uel sistema y, sobie touo, paia obtenei la
ieuuccion ue las uimensiones, ue mouo que la infoimacion ielevante
conteniua en la matiiz multiuimensional pueua queuai ieflejaua sobie
otias uos o ties uimensiones oblicuas obteniuas como combinaciones
lineales ue las vaiiables oiiginales.

El PCA es una técnica multivaiiante que peimite tiansfoimai un
giupo inicial ue vaiiables coiielacionauas en otio ue vaiiables oitogonales
uenominauas componentes piincipales (PC), ue mouo que se ieuuce el
númeio ue uatos cuanuo existe coiielacion. La iuea subyacente uel PCA es
encontiai componentes piincipales que sean combinaciones lineales ue las
vaiiables oiiginales y que peimitan uesciibii caua muestia. Puesto que las
piimeias componentes piincipales iecogen la mayoi paite ue la vaiiacion
uel conjunto ue uatos, estos se pueuen iepiesentai en uos uimensiones (las
ue sus componentes piincipales) en lugai ue utilizai las totales ue oiigen.
El PCA es una heiiamienta estauistica y ue pioyeccion valiosa que peisigue
maximizai la infoimacion ue la vaiianza piesente en una matiiz ue uatos y
iepiesentaila en el menoi númeio ue uimensiones posibles.
Natemáticamente, estas opeiaciones se consiueian tiansfoimaciones
simples ue algebia lineal, uonue los nuevos ejes ue cooiuenauas,
uenominauos "egienvectois" (vectoies piopios, o componentes piincipales)
y que son combinaciones lineales entie las vaiiables oiiginales, cumplen
Intiouuccion


S9
auemás con la conuicion ue sei oitogonales entie ellas. Los componentes
piincipales son vaiiables latentes, esto es, que mouelan las piincipales
tenuencias piesentes en la matiiz ue uatos expeiimentales. 0tio aspecto
muy impoitante a tenei en cuenta en el PCA es su capaciuau paia la
ieuuccion uel númeio ue vaiiables necesaiias paia iepiesentai el sistema,
piopoicionanuo un nuevo conjunto ue vaiiables que uesciiben
piopieuaues encubieitas hasta ese momento, ievelanuo las tenuencias
pieuominantes ue los uatos.

El análisis factoiial (FA) es una técnica multivaiiante encuauiaua
uentio uel giupo ue técnicas ue simplificacion o ieuuccion ue la uimension
y que uesue el punto ue vista estauistico no uifieie uel PCA. Sin embaigo,
uesue un punto ue vista quimico-analitico, FA auquieie una mayoi
ielevancia conceptual, ya que los factoies se consiueian entes fisicos,
mientias que los componentes piincipales son entiuaues abstiactas. La
conveision ue una entiuau abstiacta a otia ue natuialeza fisica o quimica
iecibe el nombie ue "iotacion" o "tiansfoimacion". FA es a menuuo
utilizauo paia la tiansfoimacion ue componentes piincipales "abstiactos" a
factoies quimicos bien conociuos.

Poi otia paite, los métouos ue ieconocimiento ue pautas
supeivisauos consisten en elaboiai mouelos matemáticos a paitii ue un
conjunto ue muestias que foiman paite uel conjunto ue calibiacion y que
peitenecen a uifeientes categoiias conociuas. Los mouelos estableciuos
pueuen utilizaise paia la posteiioi clasificacion ue objetos uentio ue las
clases coiiesponuientes. En estos métouos se encuentian las técnicas ue
clasificacion y las ue mouelauo, que a su vez se uiviuen en:

1) Técnicas piobabilisticas. Entie otias, cabe señalai el análisis
uisciiminante lineal (LBA) y el cuauiático (QBA), sienuo técnicas ue
clasificacion. Entie las técnicas piobabilisticas no paiamétiicas se
incluye la técnica 0NEQ (mouelo ue clases no iguales).

2) Técnicas basauas en meuiuas ue uistancias entie objetos paia la
clasificacion (K-vecinos más pioximos, KNN), entie objetos paia el
mouelauo y entie objetos y mouelos paia la clasificacion y
mouelauo (SINCA).


4u
S) Técnicas ue clasificacion basauas en la expeiiencia, en las que las
especificaciones se auquieien meuiante un pioceuimiento ue
piueba y coiieccion: ieues neuionales aitificiales (ANN) y
máquinas ue apienuizaje lineal o cuauiático.

Be touas ellas se comentaiá la técnica ue análisis lineal
uisciiminante (LBA), que es una técnica multivaiiante que tiata ue
clasificai a inuiviuuos en giupos pieviamente ueteiminauos. Los objetivos
concietos que pietenuen cubiiise con este tipo ue análisis son uos. El
piimeio ue ellos consiste en ueteiminai a paitii ue la infoimacion inicial
sobie una seiie ue inuiviuuos, ue los cuales se conoce el giupo o poblacion
a la que peitenecen, si los inuiviuuos están peifectamente uefiniuos en
funcion ue las vaiiables uel métouo. El segunuo es clasificai a inuiviuuos,
uistintos ue los manejauos en la infoimacion inicial, en uno ue los giupos
existentes.

En las últimas uécauas, la quimiometiia ha uesempeñauo una
funcion uestacaua como heiiamienta paia los estuuios uiiigiuos a la
clasificacion ue los vinos según su vaiieuau, pioceuencia geogiáfica, tipo ue
tiatamiento iecibiuo en su manipulacion y otias tecnologias, ya que
posibilita uefinii sus caiacteiisticas oiganolépticas en concoiuancia con su
composicion quimica y asi establecei esas uifeientes foimas ue
clasificacion |1S7-142j. La legislacion ha iealizauo un gian esfueizo con
iespecto al etiquetauo y uenominacion ue oiigen ue alimentos y bebiuas, en
iespuesta a las exigencias y uemanua ue los consumiuoies y la socieuau. La
mejoia en el ienuimiento economico que supone una mayoi infoimacion y
etiquetauo ue los piouuctos con iespecto a su localizacion y pioceuencia, y
la pievencion ue fiauues en el etiquetauo, piouuccion y comeicializacion
son las ties causas que han piopiciauo el uesaiiollo ue la quimiometiia en
el ámbito enologico. Touo ello ha llevauo al uesaiiollo ue metouologias que
piopoicionen caua vez más infoimacion sobie los vinos como iespuesta ue
satisfaccion ue consumiuoies, piouuctoies y exigencias legislativas. Touas
estas exigencias han hecho uel sectoi vitivinicola un ejemplo en la
aplicacion ue la quimiometiia en la mejoia ue la infoimacion analitica
sobie la composicion ue los vinos y el aseguiamiento ue la autenticiuau ue
los mismos |14S,144j.

Intiouuccion


41
Se han implementauo uifeientes técnicas quimiométiicas paia la
uisciiminacion ue los vinos en base a uistintos ciiteiios como, poi ejemplo,
la tecnologia empleaua en la vinificacion, ue acueiuo con su iegion ue
pioceuencia, tipo y vaiieuau. Se han utilizauo vaiias técnicas ue
ieconocimiento ue pautas paia la clasificacion ue los vinos meuiante
análisis ue componentes piincipales (PCA) |14S,146j, análisis
uisciiminante lineal (LBA) |147j y canonico (CBA) |148j, ieues neuionales
aitificiales |149j, K-vecinos más pioximos (KNN) |1Suj y análisis ue giupos
(CA) |1S1j. Poi otia paite, se han uesaiiollauo metouologias ue calibiacion
multivaiiante meuiante PLS |1S2,1SSj y CAINAN |1S4j. Existe también un
gian númeio ue técnicas utilizauas paia la clasificacion en giupos ue vinos
con objeto ue evaluai su autenticiuau meuiante la ueteiminacion ue
analitos volátiles, elementos tiaza y mineiales, aminoáciuos y compuestos
fenolicos.


























42






































Intiouuccion


4S
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CAPITULO1
HERRAMIENTASANALÍTICAS

Beiiamientas Analiticas

SS









El uesaiiollo ue las investigaciones iecogiuas en la piesente
Nemoiia ha siuo posible giacias al empleo ue uiveisas heiiamientas
analiticas, consiueianuo como tales touos los elementos utilizauos en
las mismas, uesue pationes, ieactivos, uisoluciones y apaiatos
empleauos, hasta la instiumentacion más compleja. En los siguientes
apaitauos ue este capitulo se enumeian uichas heiiamientas junto
con sus caiacteiisticas más ielevantes.






















Capitulo 1

S4
1. Estándaresyreactivos

Touos los ieactivos y uisolventes empleauos fueion ue caliuau
analitica. Se uetallan a continuacion los estánuaies ue los analitos,
ieactivos y uisolventes empleauos.

1.1. Estánuaies ue fenoles y otios antioxiuantes

En la siguiente tabla se ielacionan los compuestos quimicos
utilizauos como analitos en la Nemoiia, su puieza y la casa comeicial
que las ha suministiauo.

Compuesto Pureza Suministro
Aciuo ascoibico > 99,u% SIuNA
Aciuo cafeico > 98,u% SIuNA
Aciuo feiúlico > 99,u% SIuNA
Aciuo gálico 97,S -1u2,S% SIuNA
Aciuo p-cumáiico > 98,u% SIuNA
Aciuo p-hiuioxibenzoico > 97,u% SIuNA
Aciuo piotocatecuico > 97,u% SIuNA
Aciuo salicilico > 99,u% ALBRICB
Aciuo siiingico > 9S,u% FL0KA
Aciuo vainillico > 97,u% SIuNA
Butilhiuioxianisol > 98,S% SIuNA
Butilhiuioxitolueno > 99,u% SIuNA
Catequina > 99,u% SIuNA
Catecol > 99,u% SIuNA
Citiato souico > 99,u% SIuNA
ualato ue uouecilo > 99,u% SIuNA
Epicatequina > 97,u% FL0KA
Fenol - 99,u% SIuNA-ALBRICB
Bespeiiuin > 8u,u% FL0KA
Biuioquinona > 99,u% ALBRICB
Biuioxihiuioquinona > 99,u% ALBRICB
Kaempfeiol > 97,u% SIuNA
Naiingenin > 9S,u% FL0KA
Naiingin > 9S,u% FL0KA
ualato ue octilo > 99,u% SIuNA
Phloioglucinol > 99,u% ALBRICB
Pyiogalol > 99,u% SIuNA-ALBRICB
Beiiamientas Analiticas

SS
ualato ue piopilo > 98,u% SIuNA
Queicetina > 98,u% ALBRICB
Resoicinol > 99,u% SIuNA-ALBRICB
Rutina > 94,u% SIuNA
Siiingaluehiuo > 97,u% FL0KA
Tians-iesveiatiol > 99% SIuNA
vainillina > 97% ALBRICB

Paia su coiiecta conseivacion, las uisoluciones ue touos los
estánuaies se mantuvieion piotegiuas ue la luz y a una tempeiatuia
ue 4ºC. Las uisoluciones estánuaies se piepaiaion bien en meuio
acuoso o bien en el minimo volumen ue etanol necesaiio paia su
uisolucion, eniasanuo con agua hasta el volumen final. Las uifeientes
uisoluciones ue tiabajo fueion piepaiauas uiaiiamente meuiante
uilucion ue las anteiioies en agua uestilaua.


1.2. Bisolventes

Se han empleauo uiveisos uisolventes uuiante el uesaiiollo ue
las investigaciones, tales como metanol, acetonitiilo, etanol, n-hexano
etc.
Estos uisolventes se empleaion con uifeientes finaliuaues:

1) Piepaiacion ue las uisoluciones estánuaies ue los analitos.
2) Limpieza y aconuicionamiento ue las columnas
ciomatogiáficas.
S) Como componentes ue las fases moviles utilizauas en los
sistemas ciomatogiáficos.


1.S. Reactivos

Bisoluciones iegulauoias: Noimalmente ha siuo necesaiio el
ajuste uel pB ue las muestias con el objeto ue conseguii la aciuez
o basiciuau iequeiiua paia el uesaiiollo ue las ieacciones
utilizauas. Paia ello se han utilizauo uiveisas uisoluciones
Capitulo 1

S6
iegulauoias meuiante tiis(hiuioximetil)aminometano, fosfato
souico, boiato souico y acetato souico, entie otios.

Sistemas ueiivatizantes: Se han utilizauo en esta Nemoiia: nitiato
ue teibio(III) pentahiuiato (ALBRICB), oxiuo ue tii n-octilfosfina
(T0P0) (ALBRICB), áciuo etilenuiamintetiaacético (EBTA)
(SIuNA), nitiato ue aluminio(III) (SIuNA) y sulfato ue ceiio(Iv)
(NERCK).

0tios:
o Suifactantes: Tiiton X-1uu (SIuNA), souio uouecil
sulfato (SBS) (SIuNA) y Biomuio ue
hexaueciltiimetilamonio (EuA-CBENIE).
o Fluoiofoios ue laiga longituu ue onua: violeta ue
ciesilo (SIuNA) y veiue ue inuocianina (SIuNA).
o Enzima laccasa (Tiametes veisicoloi) (SIuNA).
o Aciuo tetiacloioauiico (SIuNA)

2. SistemasFIA

Paia uesaiiollai las ieacciones ueiivatizantes ue los tiabajos
iealizauos meuiante ciomatogiafia ue liquiuos, ha siuo necesaiia la
utilizacion ue sistemas FIA que peimitan la incoipoiacion ue los
ieactivos en el sistema. Con este fin se ha hecho uso ue touos
elementos citauos a continuacion:

válvula ue inyeccion Rheouyne 772Si ue seis vias (Rohneit
Paik, CA, 0SA).
Bomba peiistáltica ue baja piesion uilson (villieis-le-Bel,
Fiance) Ninipuls S.
Tubos ue PTFE ue u,S mm ue uiámetio inteino paia conuucii
las uisoluciones, asi como paia fabiicai los bucles y ieactoies
necesaiios.
Conectoies ue PTFE (0mnifit) paia unii los tubos ue
conuuccion ue los uifeientes componentes uel sistema ue flujo.



Beiiamientas Analiticas

S7
3. Instrumentación

En el uesaiiollo expeiimental ue esta Nemoiia se ha utilizauo la
siguiente instiumentacion:

Ciomatogiafo ue liquiuos Agilent 12uu compuesto poi una
bomba cuateinaiia, una uniuau uesgasificauoia, una uniuau
automuestieauoia, compaitimento paia la columna
teimostatauo y uos uetectoies: un fotometio ue uiouos en fila
(BAB) y un fluoiimetio. Se ha utilizauo una columna 0nyx
monolitica ue C
18
(Phenomenex, Toiiance, CA), ue
uimensiones 1uumm x 4,6mm y tamaño ue poio: 1Snm
(mesopoios) y 2µm (maciopoios).
Fluoiimetios: Se ha utilizauo un espectiofluoiimetio SLN
Aminco (0ibana, IL) AB2 con una lámpaia continua ue Xenon
ue 1SuW y una lámpaia pulsaua ue xenon ue 7W, con una
célula ue flujo 176-uS2-QS Bellma (Bellma Bispania,
Baicelona, Spain) ue volumen inteino ue 18µL. También se ha
empleauo un espectiofluoiimetio Caiy Eclipse vaiian (Walnut
Cieek, CA, 0SA).
Espectiofotometio 0v¡vIS Lambua SS (Peikin Elmei).
Bispositivo SPR Biosuplai 4uuT, (NIvITEC)
Nicioscopio electionico ue tiansmision Philips CN-1u,
iesolucion u.S nm × u.S4 nm y equipauo con una cámaia
uigital megaview III. Rejillas ue cobie (2uuC-FC) iecubieitas
con una pelicula ue caibon Foimvai¢ 2uumesh, suministiauo
poi Aname (Nauiiu, Spain)


4. Aparatos

Buiante el uesaiiollo ue esta Nemoiia se han empleauo los
apaiatos que se uesciiben a continuacion:
Baño teimostático BAAKE BC1 (TheimoElection, Kailsiuhe,
ueimany).
Nouulo ue flujo ueteniuo ue cinética iápiua RX-2uuu (Applieu
Photophysics, Leatheiheau, 0K).
Niciobalanza Exploiei (0BA0S)
Capitulo 1

S8
5. Programasinformáticos

Se empleaion uifeientes piogiamas infoimáticos paia el
calculo estauistico y elaboiacion ue las uifeientes
iepiesentaciones giáficas: 0iigin 7.u (0RIuIN LAB), Statgiaphics
S.1, SPSS 1S.u y Niciosoft Excel














CAPITULO 2
INNOVACIONES EN LA DETERMINACIÓN
CROMATOGRÁFICA DE ANTIOXIDANTES EN
ALIMENTOS





















































Innovaciones en la ueteiminacion ciomatogiáfica ue antioxiuantes en alimentos

61
En este capitulo se uesciiben las investigaciones iealizauas paia el
establecimiento ue ties métouos ciomatogiáficos paia la ueteiminacion ue
compuestos fenolicos en alimentos y un métouo quimiométiico paia la
clasificacion ue vinos. En concieto, estas investigaciones han uauo lugai a
los siguientes aiticulos:

- Analytical innovations in the uetection of phenolics in wines. P.
Russo, A. Anuieu-Navaiio, }.N. Feinánuez-Romeio, A. uomez-
Bens, J. Agric. Food Chem. 2uu8, S6, 18S8-186S.
- Luminescent ueteimination of flavonoius in oiange juices by LC
with post-column ueiivatization with aluminium anu teibium. A.
Anuieu-Navaiio, }.N. Feinánuez-Romeio, A. uomez-Bens, J. Sep.
Sci. 2u1u, SS, Su9-S1S.
- Long-wavelength fluoiescence uetection of flavonoius in oiange
juices by LC. A. Anuieu-Navaiio, }.N. Feinánuez-Romeio, A.
uomez-Bens, Chromatographia 2u1u, 72, 111S-112u.
- 0sefulness of teibium-sensitiseu luminescence uetection foi the
chemometiic classification of wines by theii content in phenolic
compounus. A. Anuieu-Navaiio, P. Russo, N.P. Aguilai-Caballos,
}.N. Feinánuez-Romeio, A. uomez-Bens, Food. Chem. 2u11, 124,
17SS-17S9.

El aspecto más noveuoso ue los métouos ciomatogiáficos
piopuestos se encuentia en el uesaiiollo ue nuevos sistemas ue
ueiivatizacion postcolumna que peimitan la utilizacion ue la luminiscencia
sensibilizaua ue teibio y ue la fluoiescencia ue laiga longituu ue onua como
sistemas ue ueteccion, con objeto ue mejoiai los limites ue ueteccion y, en
su caso, la selectiviuau, ue los métouos ciomatogiáficos pieviamente
uesciitos paia la ueteiminacion ue polifenoles en alimentos. En la
Intiouuccion ue esta Nemoiia se han uesciito algunas ue las piincipales
caiacteiisticas ue estos sistemas ue ueteccion, asi como su aplicabiliuau
analitica. También se han uiscutiuo aspectos básicos ue las técnicas
quimiométiicas utilizauas.

0n factoi común a los ties métouos ciomatogiáficos uesaiiollauos
ha siuo la utilizacion ue una columna monolitica, como alteinativa a las
columnas empaquetauas utilizauas convencionalmente en ciomatogiafia
ue liquiuos paia la sepaiacion ue polifenoles. A uifeiencia ue estas
Capitulo 2

62
columnas, la columna monolitica está foimaua poi un sopoite soliuo,
noimalmente ue silice, con liganuos octauecil silano enlazauos a su
supeificie. Su piincipal caiacteiistica es su estiuctuia poiosa bimoual, con
maciopoios, cuyo tamaño pueue sei ue u.S a 8 µm, y mesopoios con
tamaño ue 2 a 2u nm. Los piimeios peimiten que la fase movil pase a
velociuau ielativamente elevaua a tiavés ue la columna mientias que los
mesopoios peimiten que se piouuzca la sepaiacion ue los analitos. Se ha
uemostiauo que esta estiuctuia uisminuye los tiempos ue ietencion y
mejoia notablemente la iesolucion fiente a las columnas convencionales
|1-Sj. Aunque otia caiacteiistica ue estas columnas es la posibiliuau ue
utilizai cauuales muy elevauos, hasta ue 9 mL min
-1
, los métouos
piesentauos en este capitulo utilizan cauuales más bajos ya que ha siuo
necesaiio compatibilizai el cauual ue fase movil con el cauual uel sistema
ue ueiivatizacion paia conseguii los máximos valoies ue los picos
ciomatogiáficos. No obstante, la utilizacion ue la columna monolitica ha
peimitiuo uisminuii notablemente la uuiacion uel pioceso ciomatogiáfico,
como se uiscute posteiioimente.



Bibliogiafia

|1j B.R. Bunch, S. Wang, }. Sep. Sci., 2u11, S4, 2uuS-2u12.
|2j u. uuiochon, }. Chiomatogi. A, 2uu7, 1168, 1u1-168.
|Sj L. Rieux, B. Nieueilänuei, E. veipooite, R. Bischoff, }. Sep. Sci.,
2uuS, 28, 1628-1641.














Innovaciones en la ueteiminacion ciomatogiáfica ue antioxiuantes en alimentos

6S





}. Agi. Foou Chem. 2uu8, S6, 18S8-186S

Analytical innovations in the detection of phenolics in
wines
Pietro Russo, Álvaro Andreu-Navarro, María-Paz Aguilar-Caballos,
Juan-Manuel Fernández-Romero, Agustina Gómez-Hens
Bepaitment of Analytical Chemistiy. "Naiie Cuiie Annex" builuing. Campus
of Rabanales. 0niveisity of Coiuoba, E-14u71 Coiuoba, Spain



A liquiu chiomatogiaphic methou with on-line photometiic anu
luminescent uetection foi the ueteimination of eighteen phenolic
compounus in wines is iepoiteu. Photometiic uetection is peifoimeu at
foui wavelengths, namely 2S6, 28u, S2u anu S6S nm, using a uioue aiiay
uetection system. The luminescent uetection is achieveu by means of a
post-column ueiivatization ieaction of ten of these compounus with
teibium(III) in the piesence of syneigistic agents, such as
ethyleneuiaminetetiacetic aciu (EBTA) anu n-octyltiiphosphine oxiue
(T0P0). A micellai meuium pioviueu by the suifactants souium
uouecylsulfate anu Tiiton X-1uu was useu foi the ueteimination of the
luminescent chelates at H
ex
S17, H
em
S4S nm. The long wavelength emission
of lanthaniue chelates can minimize inteifeiences fiom backgiounu sample
matiix, which usually emit at shoitei wavelengths. The analytical featuies
of the photometiic anu the fluoiimetiic methous, such as uynamic ianges of
the calibiation giaphs, uetection limits anu piecision uata have been
obtaineu. The piactical usefulness of the uevelopeu methous is
uemonstiateu by the analysis of Spanish anu Italian wine samples (ieu,
iosé, oloioso anu white), which weie uiluteu anu uiiectly injecteu into the
chiomatogiaphic system. The accuiacy of both methous was checkeu out
by assaying a iecoveiy stuuy, which was peifoimeu at thiee uiffeient
analyte levels foi each type of sample.
Capitulo 2

64
Introduction.

Phenolics aie a wiue gioup of compounus constituteu by phenolic
aluehyues, hyuioxybenzoic anu hyuioxycinnamic acius, catechins, flavonols,
anu stilbenes, in theii monomeiic foim oi conjugateu to some species, such
as taitaiic aciu in the case of cinnamic acius (1), among otheis. These
compounus aie piesent in wines because they aie seconuaiy metabolites of
plants. The composition of phenolics anu theii concentiation uepenu on
giape vaiiety, geogiaphical oiigin, soil type, collection system, anu giape
piocessing. These compounus aie iesponsible of the sensoiy piopeities of
the wines, anu, also, they aie anti-caicinogenic anu have an anti-
inflammatoiy action when they aie iegulaily ingesteu. A paiticulai
example of the impoitance of monitoiing phenolic concentiation to contiol
the quality of wines is the piesence of aiomatic aluehyues, foimeu by a
gioup of volatile compounus that aie extiacteu fiom woou lignin uuiing the
winemaking piocess. The piesence of these aluehyues in wines is an
inuicatoi of feimentation anu aging in oak baiiels, theii absence being
inuicative of counteifeit ageu wines. vanillin anu syiingaluehyue aie the
most abunuant aiomatic aluehyues in wines.

The occuiience of phenolics has been extensively stuuieu by liquiu
chiomatogiaphic methous (1–18). In most of them, conventional ieveiseu-
phase columns, constituteu by packeu miciopaiticulate bonueu silica, have
been useu (1–5, 7–15, 18), which geneially featuie sepaiations of 14-2S
compounus in almost 1h oi S2 phenolics in 9u min, which makes ioutine
analysis of these compounus veiy teuious. The use of othei mateiials such
as mesopoious silica has given iise to monolithic columns, which opeiate at
highei flow iates with lowei back- piessuies than conventional columns
(16, 17). Thus, they allow the analysis of samples using uiiect injection oi
low sample uilutions, because the cleaning anu iegeneiation of the column
can be uone moie quickly than in the conventional ones uue to the high
flow iates affoiueu. Nonolithic columns have been useu foi the
ueteimination of 1S phenolics in ieu anu white wine samples (16) with
sepaiation times aiounu Su min anu, also, foi the uiiect analysis of ciuei
samples (19).

Bioue aiiay uetection (1, 3–17) has been extensively useu foi the
uevelopment of liquiu chiomatogiaphy methous, wheieas fluoiometiic (8,
Innovaciones en la ueteiminacion ciomatogiáfica ue antioxiuantes en alimentos

6S
12) anu mass spectiometiy (2) uetection systems have been useu to a
lessei extent. Nost fluoiometiic methous pioposeu aie baseu on
measuiements of the intiinsic fluoiescence of some phenolics. Although
these methous featuie geneially lowei uetection limits than photometiic
methous, the numbei of compounus ueteimineu by measuiing theii native
fluoiescence is low (8, 12). An appioach that has been uesciibeu iecently is
the use of a postcolumn ueiivatization ieaction with teibium(III) foi the
ueteimination of eight compounus, namely, hyuioxybenzoic acius anu
catechins (18). The methou involves the foimation of luminescent chelates
between phenolics anu the lanthaniue ion in an alkaline meuium in the
piesence of ethyleneuiaminetetiaacetic aciu (EBTA) to pievent teibium
piecipitation. The uetection limits achieveu weie lowei than oi compaiable
to those iepoiteu by othei methous (1, 3–11, 13–17).

The woik piesenteu heie iepoits the ueteimination of 18 phenolics,
which incluue hyuioxybenzoic (gallic, piotocatechuic, p-hyuioxybenzoic,
salicylic, vanillic, anu syiingic) anu hyuioxycinnamic (caffeic, feiulic, p-
coumaiic) acius, phenolic aluehyues (syiingaluehyue, vanillin), catechins
(catechin, epicatechin), flavonols (iutin, queicetin, kaempfeiol), anu
stilbenes (cis- anu trans-iesveiatiol). The sepaiation is achieveu in <2S min
using a monolithic column. Bioue aiiay (LC-BAB) anu luminescence (LC-
FL) uetection systems aie useu simultaneously to uetect anu quantify these
phenolics. The luminescent uetection is baseu on the ieaction of
teibium(III) with 1u of these phenolics to give iise to luminescent chelates
at a slightly basic meuium, using tii n-octylphosphine oxiue (T0P0) anu
EBTA as syneigistic agents. Byuioxybenzoic acius, catechins, anu
aluehyues aie uetecteu by means of this ueiivatization ieaction. Teibium-
sensitizeu luminescence is useu foi the fiist time to ueteimine aiomatic
aluehyues. The luminescence of the chelates is piotecteu fiom non
iauiative piocesses by a micellai meuium pioviueu by Tiiton X-1uu anu
souium uouecyl sulfate. The photometiic uetection is accomplisheu at foui
wavelengths: 2S6, 28u, S24, anu S6S nm, wheieas the luminescent
uetection is peifoimeu using S17 anu S4S nm as excitation anu emission
wavelengths, iespectively. Both uetection systems aie complementaiy
tools to iuentify anu quantify phenolic compounus in uiffeient kinus of
wine samples, such as ieu, iosé, oloioso, anu white. The only tieatment
neeueu is sample uilution piioi to the injection onto the chiomatogiaphic
system. This tieatment is simplei than those uesciibeu elsewheie (1, 3, 8,
Capitulo 2

66
10, 12, 14), which involve the extiaction anu¡oi fiactionation of phenolics.
The impioveu selectivity of sensitizeu luminescence can facilitate the
iuentification of some compounus that cannot be easily iuentifieu using 0v
uetection in the piesence of complex wine samples. The analytical featuies
of both methous as well as theii peifoimance in the analysis of wine
samples aie compaieu.


1. Materials and methods

1.1. Appaiatus anu Instiuments.

An Agilent 12uu seiies liquiu chiomatogiaphy system composeu of
a quateinaiy pump, a uegassei unit, a vial autosamplei, a theimostateu
column compaitment, anu a uioue aiiay uetectoi was useu. An SLN Aminco
(0ibana, IL) AB2 luminescence spectiometei pioviueu with a 1Su W
continuous xenon lamp anu a 7 W pulseu xenon lamp, fuinisheu with a
176-uS2-QS Bellma (Bellma Bispania, Baicelona, Spain) flow cell with an
innei volume of 18 µL, was useu to monitoi fluoiescence measuiements. A
uilson (villieis-le-Bel, Fiance) Ninipuls S low-piessuie peiistaltic pump
anu 0mnifit (Cambiiuge, 0.K.) Teflon tubing of u.S mm i.u. weie also useu
foi constiucting the postcolumn ueiivatization manifolu. Chiomatogiaphic
sepaiation was peifoimeu using an 0nyx monolithic C18 column
(Phenomenex, Toiiance, CA), 1uu mm × 4.6 mm i.u.; poie sizes weie
mesopoies (1S nm) anu maciopoies (2 µm).


1.2. Reagents.

All chemicals useu weie of analytical ieagent giaue. Stock solutions
(Suuu mg L
-1
) of phenolics weie piepaieu as follows: phenolic acius, such
as vanillic, gallic, piotocatechuic, p-hyuioxybenzoic (Sigma), syiingic
(Fluka), anu salicylic (Aluiich) acius, weie piepaieu by uissolving them in a
minimum volume of ethanol (1u-2S mL, uepenuing upon the phenolic
consiueieu) anu biinging them up to the final volume (Su mL) with
uistilleu watei. The same pioceuuie was useu foi hyuioxycinnamic acius,
such as feiulic, caffeic, anu p-coumaiic (Sigma) acius; the aiomatic
aluehyues vanillin (Aluiich) anu syiingaluehyue (Fluka); the catechins
Innovaciones en la ueteiminacion ciomatogiáfica ue antioxiuantes en alimentos

67
catechin anu epicatechin, anu, also, iesveiatiol (Sigma), all of which weie
uegasseu using nitiogen to pievent theii oxiuation by uissolveu oxygen.
Queicetin (Aluiich), iutin (Sigma), anu kaempfeiol (Sigma) weie uissolveu
in absolute ethanol. cis-Resveiatiol was obtaineu by iiiauiating an aliquot
of trans- iesveiatiol stock solution with a 0v lamp at S6u nm foi 2 h at
ioom tempeiatuie. The obseiveu yielu foi this change was calculateu by
taking into account the ueciease in the aiea of the peak of trans-iesveiatiol
at S24 nm. Fifty peicent of trans-iesveiatiol was conveiteu unuei the
mentioneu iiiauiation conuitions. Inteimeuiate solutions of 1uu mg L
-1

weie piepaieu by uiluting the stock solutions in uistilleu watei, except foi
queicetin, iutin, iesveiatiol, anu kaempfeiol, which iequiieu absolute
ethanol to be stable. Stock anu inteimeuiate solutions weie stoieu at 4 °C in
the uaik anu weie stable foi at least 2 weeks. Woiking stanuaiu solutions
weie piepaieu fiom inteimeuiate solutions by theii uilution in uistilleu
watei.

The mobile phase useu foi the sepaiation was constituteu by
solvent A (acetic aciu u.u2%, pB aujusteu to S.8S with souium hyuioxiue,
Panieac Quimica, S.A., Baicelona, Spain) anu solvent B |BPLC-giaue puie
acetonitiile (ACN), Panieac Quimica, S.A.j, which weie mixeu by opeiating
in the giauient moue uuiing the chiomatogiaphic sepaiation. The
ueiivatization ieagent was foimeu in situ by mixing two stieams: the fiist
one containeu a solution of teibium(III) (7.S × 1u
-S
N), piepaieu fiom
teibium(III) nitiate pentahyuiate (Aluiich), anu the seconu one, a
continuously stiiieu mixtuie integiateu by T0P0 (Sigma) (7 × 1u
-4
N),
Tiiton X-1uu (Fluka) (u.8%), EBTA (Fluka) (1.2 × 1u
-S
N),
souiumuouecylsulfate (Neick) (1u
-S
N), anu
tiis(hyuioxymethyl)aminomethane (Neick) buffei (Tiis) (u.2 N, pB 9.S).


1.3. Nanifolu anu Pioceuuie.

Figure 1 shows the thiee-step integiateu LC
sepaiation¡ueiivatization¡uetection appioach. Stanuaius oi uiluteu
samples (Su µL), containing the analytes at concentiations within theii
coiiesponuing uynamic ianges, weie injecteu into the column. The mobile
phase was pumpeu at 2 mL min-1, anu the system opeiateu unuei the
giauient conuitions incluueu in Table 1. The vaiiation of absoibance with
Capitulo 2

68
time was monitoieu at 2S6, 28u, S2u, anu S6S nm. The time necessaiy to
achieve a LC-BAB chiomatogiam was 2S min. Then, a cleanup anu
conuitioning step weie applieu to have the chiomatogiaphic system ieauy
foi the next injection aftei S min.

The eluate of the column aftei passing thiough the uioue aiiay
uetection system was meigeu at point u with the ueiivatizing solution,
which was pumpeu at a flow iate of u.8 mL min
-1
. The mixeu solution
passeu thiough the ieactoi L1, in which the ueiivatization ieaction took
place. The fluoiescence intensity was monitoieu at iex S17 anu iem S4S
nm foi 2u min, anu the coiiesponuing blank solutions weie subtiacteu.
Chiomatogiams weie taken using the oiiginal softwaie of the luminescence
spectiometei, anu the iaw uata of luminescence intensity anu time weie
expoiteu anu tieateu using auequate softwaie packages foi the estimation
of the main chiomatogiaphic paiameteis.


















Figure 1. Integiateu sepaiation-ueiivatization anu uetection appioach. 1, 2 anu S uepict the
chiomatogiaphic, ueiivatizing anu uetection subsystems, iespectively. A, anu B,
uenotes u.u2% acetic aciu anu acetonitiile solutions, iespectively; SBS, solvent
ueliveiy system; BPP, Bigh-piessuie quateinaiy giauient pump; AS0, autosamplei
unit; C18 NC, C18 monolithic column; TCC, theimostateu column compaitment; BAB
uioue aiiay uetection system; R1 anu R2, ieagent stieams 1 anu 2; LPP, low-
piessuie pump; L1, mixing ieactoi; FLB, fluoiescence uetectoi; PC, peisonal
computei; w1, waste.


Innovaciones en la ueteiminacion ciomatogiáfica ue antioxiuantes en alimentos

69
Table 1. uiauient elution conuitions
Time (min) %A %B Flow (ml min
-1
)
u 2.u 98.u 2.u
7.uu 2.u 98.u 2.u
9.uu 6.u 94.u 2.u
14.uu 6.u 94.u 2.u
19.uu 16.u 84.u 2.u
2u.uu 16.u 84.u 2.u
22.uu Su.u 7u.u 2.u
24.uu Su.u 7u.u 2.u
2S.uu 2.u 98.u 2.u
Conditioning step
2S.Su 8u.u 2u.u 2.S
27.uu 8u.u 2u.u 2.S
27.1u 2.u 98.u 2.u
Su.u 2.u 98.u 2.u
A: acetonitiile, B: acetic aciu (u.u2 %, pB S.8S)



1.4. Assessment of Piecision.

Piecision was evaluateu at two uiffeient concentiation levels, 2uu
anu 6uu ng mL
-1
(except foi cis- anu trans- iesveiatiol, which weie 1uu
anu Suu ng mL
-1
anu Suu anu 9uu ng mL
-1
, iespectively). Seven solutions
weie subjecteu to the chiomatogiaphic sepaiation on the same uay anu 1u
solutions maue in uuplicate on five uiffeient uays to calculate in-uay anu
inteiuay, iespectively, piecision foi ietention times anu aieas by obtaining
the peicentage of ielative stanuaiu ueviation of these paiameteis in each
case.

1.5. Estimation of LODs.

The estimation of L0Bs was uone following I0PAC
iecommenuations (20), which involve the use of a signal-to-noise iatio of S.
The signal consiueieu as blank signal was the stanuaiu ueviation of the y-
inteicept of the calibiation cuive maue using 1u mixtuies of aqueous
stanuaius of the phenolics ueteimineu.


Capitulo 2

7u
1.6. Analysis of Wine Samples.

Wine samples weie uiluteu with uistilleu watei to match the lineai
ianges of calibiation foi each analyte anu weie uiiectly injecteu onto the
chiomatogiaphic system following the pioceuuie above inuicateu. Each
ueteimination was the mean of thiee measuiements.


2. Results and discussion

2.1. Stuuy of Bioue Aiiay anu Luminescent Betection Systems.

Absoibance spectia weie iecoiueu in the iange of 2uu-4uu nm foi
selecting the wavelengths of the LC-BAB methou. Foui wavelengths (2S6,
28u, S24, anu S6S nm), which aie close to the maximum absoiption
wavelength of each phenolic, weie chosen.

Fluoiometiic uetection was achieveu using teibium(III) as
ueiivatizing ieagent. The ielatively long emission wavelength of
teibium(III) chelates can impiove the selectivity anu sensitivity of the
measuiements because some fluoiescent signals fiom sample matiix,
which coulu oveilap the chiomatogiaphic peaks of the phenolics, aie
minimizeu oi avoiueu. Postcolumn ueiivatization with teibium(III), using
an alkaline meuium anu in the piesence of EBTA, has been pieviously
iepoiteu foi the ueteimination of some hyuioxybenzoic acius anu
catechins in white wine samples (18). Bowevei, aiomatic aluehyues uo not
give any luminescent signal unuei these expeiimental conuitions. It has
been necessaiy to mouify this ueiivatization ieaction to obtain luminescent
teibium(III) chelates of aiomatic aluehyues. These chelates aie foimeu in a
slightly basic meuium, pioviueu by a Tiis buffei solution, anu using T0P0
anu EBTA as syneigistic agents. Byuioxybenzoic acius anu catechins also
give luminescent signals unuei these conuitions. Table 2 shows the
maximum absoiption wavelengths of phenolics, which weie useu to
establish the LC-BAB methou. Some of these compounus also piesent othei
less intense absoiption wavelengths, which appeai in this table as
confiimation wavelengths. These wavelengths can be chosen as an
alteinative when an impiovement of the selectivity is iequiieu uue to
chiomatogiaphic oveilapping of othei peaks at the maximum absoiption
Innovaciones en la ueteiminacion ciomatogiáfica ue antioxiuantes en alimentos

71
wavelength, although the signal obtaineu is lowei. Table 2 also shows the
excitation anu emission wavelengths coiiesponuing to 1u of the phenolics
assayeu, which foim luminescent chelates with teibium(III). Although they
show uiffeient maximum excitation wavelengths, theii bioau excitation
banus allow the selection of S17 nm as the woiking wavelength foi the
simultaneous excitation of these chelates. This wavelength was chosen to
get the maximum sensitivity foi catechin, vanillin, syiingaluehyue, anu
epicatechin, which exhibiteu lowei luminescence intensities than
hyuioxybenzoic acius unuei the expeiimental conuitions assayeu. The
luminescence emission is measuieu at S4S nm, which is the most intense
emission wavelength of teibium(III).

Table 2. Chaiacteiistic wavelengths of the stuuieu compounus

Compounu
LC-BAB
LC-FL
Phenolic-teibium chelate
Naximum
absoiption
wavelength
(nm)

Confiimation
wavelength
(nm)
Naximum
excitation
wavelength
(nm)
Emission
wavelength
(nm)

uallic aciu 28u 2S6 S17
S4S
Piotocatechuic aciu 2S6 28u S1S
p-Byuioxy benzoic
aciu
2S6 28u 24S, 299
Salicylic aciu 28u --- 29S
vanillic aciu 2S6 28u 298
Caffeic aciu S24 28u ---
Syiingic aciu 28u 2S6 29S
Catechin 28u --- Su2
vanillin 28u 2S6, S24 S24
p-Coumaiic aciu 28u S24 ---
Syiingaluehyue S24 28u SSS
Epicatechin 28u 2S6, S24 299
Feiulic aciu S24 28u ---
Rutin 2S6 S6S ---
tians-Resveiatiol S24 --- ---
Queicetin S6S --- ---
cis-Resveiatiol 28u --- ---
Kaempfeiol S6S --- ---
Capitulo 2

72
2.2. 0ptimization of vaiiables.

The hyuiouynamic anu chemical vaiiables involveu in LC-BAB anu
LC-FL methous weie optimizeu using the univaiiate methouology. values
chosen weie those yieluing the maximum absoibance anu luminescence
signals with a minimum stanuaiu ueviation.


2.3. Chiomatogiaphic vaiiables.

The luminescence of lanthaniue chelates can be quencheu by the
vibiation of hyuioxyl gioups of watei molecules (21), which is ieally
notable when RP-LC is useu uue to the ielatively high content of watei in
the mobile phases useu. The auuition of a syneigistic agent oi the
intiouuction of an aciu in the mobile phase can help to achieve the iequiieu
chiomatogiaphic sensitivity. It has been pieviously uesciibeu that an
auequate luminescent signal can be obtaineu in the piesence of acetate ions
(22, 23), which can be asciibeu to theii capability to uisplace watei fiom
the fiist cooiuination spheie of teibium(III) (24).

Chiomatogiaphic vaiiables weie optimizeu to sepaiate the analytes
in the shoitest sepaiation time. The stuuy of the effect of the appaient pB
of the mobile phase showeu that it is a ciitical vaiiable to achieve the
iequiieu chiomatogiaphic selectivity. The shape of peaks was moie
uefinite at pB S.8S, using u.u2% acetic aciu. The pB of the mobile phase was
ciitical foi the sepaiation of the peaks of caffeic anu syiingic acius anu the
peaks of syiingaluehyue anu epicatechin. Binaiy mixtuies of acetic aciu
(u.u2%, pB S.8S) anu ACN using uiffeient giauient piofiles weie tiieu at
the optimum flow iate (2 mL min-1), finuing that the use of incieasing
peicentages of ACN in two subsequent steps (Table 1), fiom 14 to 19 min
anu fiom 2u to 22 min, was useful foi the iesolution of p-coumaiic,
syiingaluehyue, epicatechin, anu feiulic aciu. Initial peicentages of ACN
>2% alloweu lowei ietention times foi all of the compounus, but they weie
oveilapeu.

The optimum conuitions founu weie those appeaiing in Table 1,
which weie useu foi the chiomatogiaphic sepaiation. As can be also seen
fiom this table, cleaning anu conuitioning steps, which take only S min, aie
Innovaciones en la ueteiminacion ciomatogiáfica ue antioxiuantes en alimentos

7S
incluueu in the giauient ioutine to ensuie iepiouucibility of ietention
times between two iepeateu injections.

2.4. Postcolumn Beiivatization vaiiables.

The stuuy of the flow iate of the ueiivatizing ieagent solution
showeu that u.8 mL min
-1
pioviueu the optimum ueiivatizing solution
column effluent iatio when 2 mL min
-1
was useu foi the chiomatogiaphic
sepaiation. The length of the ieactoi L1 useu was Su cm, which is enough
foi the uevelopment of the ueiivatization ieactions uue to the ielatively
fast iate foi the teibium chelate foimation. A Tiis buffei solution was
chosen to inciease the pB of the chiomatogiaphic eluent, which is iequiieu
to achieve the optimum luminescence signal without teibium hyuioxiue
piecipitation. The concentiation of this buffei was stuuieu in the iange of
u.uS-u.2 N, the pB being aujusteu to 8.7 using the highest concentiation.
The influence of teibium(III) concentiation was evaluateu foi each
phenolic-teibium chelate in the iange of S×1u
-4
- 1.u×1u
-2
N (Figure 2).

The concentiation of T0P0 was stuuieu in the iange of 1.S×1u
-4
-
1.6×1u
-S
N. This syneigistic agent notably enhances the sensitizeu
luminescence of salicylic aciu anu catechin when it is useu at (2-7.S) × 1u
-4

N. This behavioi was less significant foi the othei phenolics assayeu. EBTA
was mainly useu to pievent the teibium piecipitation, anu its influence was
stuuieu in the iange of 2×1u
-4
- 1.u×1u
-2
N. It was founu that this compounu
impioves the signal of gallic aciu, but uecieases the signal fiom the othei
compounus. Thus, a compiomise solution was taken by using 1.2×1u
-S
N
EBTA. The suifactants Tiiton X-1uu anu souium uouecyl sulfate weie useu
to piotect the luminescence of the chelates fiom noniauiative piocesses.
Tiiton X-1uu hau a positive influence on the signal of gallic aciu, although
the luminescence fiom othei phenolic chelates was unalteieu until u.8%,
fiom which a ueciease was obseiveu, this concentiation being chosen as
optimum. The othei suifactant incieases the signal of syiingaluehyue,
which was piactically negligible in its absence. Figures 3 anu 4 uepict the
chiomatogiams obtaineu foi a stanuaiu mixtuie of phenolics, caiiieu out
unuei optimum conuitions using LC-BAB anu LC-FL methous, iespectively.
As can be seen fiom Figure 3, the highest numbei of analytes appeais in
the chiomatogiam obtaineu at 28u nm.

Capitulo 2

74
[Tb(III)], M
0.000 0.002 0.004 0.006 0.008 0.010
Area
0
20
40
60
80
100
2
1
3
9
5
4
7
11
8,12
which was piactically negligible in its absence. Figures 3 anu 4 uepict the
chiomatogiams obtaineu foi a stanuaiu mixtuie of phenolics, caiiieu out
unuei optimum conuitions using LC-BAB anu LC-FL methous, iespectively.
As can be seen fiom Figure 3, the highest numbei of analytes appeais in
the chiomatogiam obtaineu at 28u nm.





Figure 2. Influence of the teibium (III) concentiation foi (6uu ng mL
-1
): 1 gallic
aciu; 2, piotocatechuic aciu; S, p-hyuioxybenzoic aciu; 4, salicylic aciu;
S, vanillic aciu; 7, syiingic aciu; 8, catechin; 9, vanillin; 11,
syiingaluehyue; 12, epicatechin, unuei the optimum expeiimental
conuitions.

Innovaciones en la ueteiminacion ciomatogiáfica ue antioxiuantes en alimentos

7S































Figure 3. Typical chiomatogiams achieveu using the LC-BAB methou at 2S6, 28u,
S24 anu S6S nm, by injection of a mixtuie of a aqueous stanuaiu solution
containing 6uu ng mL
-1
of each analyte (excepting cis- anu tians-
iesveiatiol, which weie 9uu anu Suu ng mL
-1
)anu piocesseu unuei the
optimum conuitions uesciibeu in the Nanifolu anu Pioceuuie section. 1
gallic aciu; 2, piotocatechuic aciu; S, p-hyuioxybenzoic aciu; 4, salicylic
aciu; S, vanillic aciu; 6, caffeic aciu; 7, syiingic aciu; 8, catechin; 9,
vanillin; 1u, p-coumaiic aciu; 11, syiingaluehyue; 12, epicatechin; 1S,
feiulic aciu; 14, iutin; 1S, tians-iesveiatiol; 16, cis-iesveiatiol; 17,
queicetin; 18, kaempfeiol.
Capitulo 2

76
Time (min)
0 5 10 15 20
I
L
0
3
6
9
1
2
3
4
5
7
8 9
11
12












Figure 4. Chiomatogiam achieveu using the LC-FL methou by injection of a
mixtuie of aqueous stanuaius at 6uu ng mL
-1
of each analyte anu
piocesseu unuei the optimum expeiimental conuitions. 1 gallic aciu; 2,
piotocatechuic aciu; S, p-hyuioxybenzoic aciu; 4, salicylic aciu; S, vanillic
aciu; 7, syiingic aciu; 8, catechin; 9, vanillin; 11, syiingaluehyue; 12,
epicatechin.


2.5. Analytical Featuies.

Calibiation giaphs weie iun unuei the optimum expeiimental
conuitions by using an exteinal calibiation methou. Table 3 shows the
ietention time foi each phenolic, the calibiation paiameteis, anu the
uetection limits (L0Bs) obtaineu foi both methous, which weie calculateu
accoiuing I0PAC iecommenuations (20). These uetection limits aie lowei
than oi compaiable to those of othei methous pieviously iepoiteu (1–17,
19). Foi LC-BAB, this fact can be explaineu by two ieasons: (1) the use of a
monolithic column, which pioviues naiiowei peaks than the paiticulate
columns, enhancing the peak height anu impioving the signal-to-noise
iatio; (2) a highei injection volume (Su µL) than those useu in the othei
methous, which aie typically 1u-2u µL. This injection volume uoes not
pioviue notable peak bioauening uue to the ielatively high flow iate useu
in the piesent methou. The r2 values obtaineu inuicate a veiy goou
coiielation of expeiimental uata to calibiation cuives. The piecision of
both methous has been stuuieu foi ietention times anu aieas at two
uiffeient analyte concentiation levels, anu the iesults obtaineu at 6uu ng
mL
-1
foi most analytes, expiesseu as the peicentage of ielative stanuaiu
ueviation, aie summaiizeu in Table 4. It can be seen that the iepeatability
of peak aieas foi both methous was equal to oi lowei than 6%, the iesults
foi the LC-FL methou being slightly highei than those obtaineu by applying
Innovaciones en la ueteiminacion ciomatogiáfica ue antioxiuantes en alimentos

77
the LC-BAB methou. The piecision was evaluateu also at 2uu ng mL
-1
,
finuing that the iesults obtaineu iangeu fiom u.1 to 1.S% anu fiom u.S to
4.S% foi the in-uay anu inteiuay, iespectively, piecisions of ietention times
anu fiom 1.2 to 7.4% anu fiom S.2 to 9.7% foi the in-uay anu inteiuay,
iespectively, piecisions of aieas obtaineu using the FL methou. Foi the BAB
methou, the iesults iangeu fiom u.u7 to 1.S% anu fiom u.1S to S% foi the
in-uay anu inteiuay, iespectively, piecisions of ietention times anu fiom
u.S to 7.S% anu fiom 1.S to 1u.7% foi the in- uay anu inteiuay, iespectively,
piecisions of aieas. This uiffeience coulu be explaineu by beaiing in minu
that LC-FL can be affecteu by moie souices of vaiiability of the iesults, such
as the use of a seconu pump anu the ueiivatization ieaction.




T
a
b
l
e

3
.

A
n
a
l
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t
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c
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f
e
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)

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l
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t

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B
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1
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6
6
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9
u
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u
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4
2

±

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u
u
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9

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7

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9
9
9
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1
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7
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1
0
-
9
0
0
0
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0
6
4
5

±

0
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0
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0
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5

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1

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9
9
9
9
5
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9
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9
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6

3
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8

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1
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6
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2
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0
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9
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±

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1
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7
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5

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9
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1
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1
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9
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u
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±

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±

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1
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6

±

u
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2

u
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±

u
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u
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9
9
9
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1
7
.
3
5
5
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0
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2
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±

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2
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u
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1

±

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.
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4

-
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2
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±

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u
.
9
9
9
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8

1
7
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5
1
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2
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0
0
.
0
0
3
6
1

±

0
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0
0
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4

0
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0
3

±

0
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0
3

0
.
9
9
8
6
2
9
F
e
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a
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1
8
.
4
2
2
u
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u
u
u
u
.
1
u
4
9
8

±

u
.
u
u
u
u
4

-
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.
4

±

u
.
2

u
.
9
9
9
9
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2
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.
8
6
1
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2
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u
u
u
.
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2
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6
4

±

u
.
u
u
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-
u
.
7

±

u
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2

u
.
9
9
9
9
2
4





T
a
b
l
e

3
.

C
o
n
t
i
n
u
a
t
i
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n

C
o
m
p
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(
m
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(
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g

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1
)

S
l
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p
e

±

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B


y
-
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n
t
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c
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p
t

±

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B
i
2
L
0
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(
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g

m
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1
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2
2
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1
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7
8
2

±

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.
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.
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8

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9
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2
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9
7

±

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.
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1

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±

2

u
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9
9
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6
u
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2
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9
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±

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±

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u
.
9
9
9
9
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2
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.
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8
9
2

±

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1

±

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u
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9
9
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T
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4
.

P
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c
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s
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n

(
%
R
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B
)

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Innovaciones en la ueteiminacion ciomatogiáfica ue antioxiuantes en alimentos
81
2.6. Applications.

LC-BAB anu LC-FL methous weie applieu to the analysis of 1S
wine samples belonging to uiffeient wine vaiieties (ieu, oloioso, iosé,
anu white). Samples W1-WS weie ieu wines, W6 was iosé wine, W7-
W1u incluueu oloioso wines, anu W11-W1S weie white wine
samples. These wine samples belongeu to Coiuoba (Spain) (samples
W1, WS, W4, W6, W1u, W11, W14, anu W1S) anu Sicily (Italy)
(samples W2, WS, W7-W9, W12, anu W1S) geogiaphical aieas. Wine
samples weie uiluteu with uistilleu watei to match the lineai iange of
the calibiation giaph foi each analyte. Figures 5 anu 6 coiiesponu to
the typical chiomatogiams achieveu foi a ieu wine sample using LC-
BAB anu LC-FL methous, iespectively.

























Time (min)
0 5 10 15 20 25 30
mAU
0
10
20
30
11
6
13
15
324 nm
Time (min)
0 5 10 15 20 25 30
mAU
0
40
80
120
160
280 nm
1
7
89
10
12
Time (min)
0 5 10 15 20 25 30
mAU
0
40
80
120
256 nm
2
3
5
14
Cápitulo 2

82










Figure 5. Chiomatogiams of a ieu wine sample (Sample W2, uilution 1¡1u)
using the LC-BAB methou obtaineu by uiiect injection of Su µL of
uiluteu sample. 1 gallic aciu; 2, piotocatechuic aciu; S, p-
hyuioxybenzoic aciu; 4, salicylic aciu; S, vanillic aciu; 6, caffeic
aciu; 7, syiingic aciu; 8, catechin; 9, vanillin; 1u, p-coumaiic aciu;
11, syiingaluehyue; 12, epicatechin; 1S, feiulic aciu; 14, iutin; 1S,
tians-iesveiatiol; 16, queicetin; 17, cis-iesveiatiol; 18,
kaempfeiol.


As can be seen fiom Figure 5, theie is an inciease in the
baseline of the chiomatogiams in the iange of 22-24 min. It has been
iepoiteu that this baseline uiift can be asciibeu to the piesence of
polymeiic compounus, which have not been sepaiateu in the
expeiimental conuitions (1). Bowevei, the chiomatogiam
coiiesponuing to the LC-FL methou (Figure 6) is cleanei than that
obtaineu foi the LC-BAB methou. This uemonstiates the highei
selectivity level obtaineu using teibium-sensitizeu luminescence,
which is less pione to baseline uiifts anu to the inteifeience fiom
othei sample components. An example of the complementaiity of the
infoimation supplieu by both LC-BAB anu LC-FL methous in the
analysis of ieal samples is the ueteimination of salicylic aciu, which
cannot be ueteimineu with accuiacy in some wine samples using
photometiic measuiements, uue to the piesence of an oveilapping
peak at the same ietention time. Bowevei, this inteifeience is not
obseiveu foi the LC-FL methou, which also gives satisfactoiy iesults
when stanuaius of salicylic aciu aie auueu. Thus, the LC-FL methou is
suitable foi the iuentification anu confiimation of some of the peaks
founu by LC-BAB.
Time (min)
0 5 10 15 20 25 30
mAU
-5
0
5
10
15
20
16
18
365 nm
Innovaciones en la ueteiminacion ciomatogiáfica ue antioxiuantes en alimentos
8S












Figure 6. Chiomatogiam of a ieu wine sample (Sample W2, uilution 1¡1u)
using the LC-FL methou obtaineu by uiiect injection of Su µL of the
sample. 1 gallic aciu; 2, piotocatechuic aciu; S, p-hyuioxybenzoic
aciu; 4, salicylic aciu; S, vanillic aciu; 7, syiingic aciu; 8, catechin; 9,
vanillin; 11, syiingaluehyue; 12, epicatechin.


Tables 5 anu 6 summaiize the phenolic content founu foi the
Spanish anu Italian wines analyzeu, iespectively; it can be seen that
the highest values weie founu in ieu wine samples. The phenolics that
foim luminescent chelates with teibium(III) weie ueteimineu using
both LC-BAB anu LC-FL methous, anu the iesults obtaineu by both
methous weie statistically compaieu using a iegiession test. The
iegiession stuuy caiiieu out with the concentiation values obtaineu
by both methous showeu that they uiu not uiffei significantly taking
into account the coiielation paiameteis obtaineu (Y = u.18S + u.979X,
r = u.9S4, Y anu X being the iesults foi LC-FL anu LC-BAB,
iespectively). cis-Resveiatiol was not founu in any of the samples.
Sicilian ieu wines contain high concentiations of gallic aciu, which
agiee with the values founu in the liteiatuie (2). Piotocatechuic anu
syiingic acius aie the most abunuant phenolics founu in white wine
samples.
Time (min)
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Capitulo 2



86
The iecoveiy stuuy was caiiieu out by auuing thiee uiffeient
amounts of each analyte to foui of the samples analyzeu at
concentiations in the iange of u.S-2u mg L
-1
anu subtiacting the
iesults obtaineu fiom similaily unspikeu samples. Table 7 shows the
mean iecoveiy values obtaineu, which weie in the iange of 68.u-
116.9%, most of them being >8S%, except those foi iutin in sample
W1 anu syiingaluehyue in sample W11, which gave values of 68%,
although the values obtaineu foi the othei analyzeu samples weie
closei to 1uu%. Also, the LC- BAB methou gave iesults similai to
those of the LC-FL methou. The iecoveiies obtaineu foi the iest of
compounus weie in agieement with those pioviueu by othei methous
(3, 6, 13, 14, 16). In some of these methous, the accuiacy was
ueteimineu using stanuaius oi synthetic wine samples (6, 13, 14) anu,
theiefoie, the iesults cannot be uiiectly compaieu with those
obtaineu with the pioposeu methou, which weie obtaineu in the
piesence of commeicial wine samples. Bespite this fact, the iesults
obtaineu weie closei to 1uu% than those pioviueu by othei methous
involving seveial extiaction steps (3).

This stuuy shows the usefulness of teibium-sensitizeu
luminescence as a complementaiy tool foi the iuentification anu
quantification of phenolic compounus in complex wine samples. This
is the fiist time that aiomatic aluehyues have been ueteimineu using
teibium-sensitizeu luminescence. The spectial selectivity achieveu
using this appioach can be piofiteu to avoiu oi minimize the potential
inteifeiences fiom the sample matiix. The sensitivity levels achieveu
with both LC-BAB anu LC-FL methous allow highei uilution of
samples to be uone, which is useful to impiove the selectivity. The
piactical application of both methous has been shown by the analysis
of 1S wine samples with satisfactoiy iesults.








Innovaciones en la ueteiminacion ciomatogiáfica ue antioxiuantes en alimentos



87

Table 7. Nean iecoveiies obtaineu by the analysis of uiffeient wine
samples

Recoveiy
(%)
Compounu W1 W2 W7 W11
uallic aciu* 1u2.S 9S.8 1u2.9 94
Piotocatechuic aciu* 92.6 9u.6 94.u 81.S
p-hyuioxy benzoic aciu* 99.8 92.7 98.4 92.1
Salicylic aciu* 1u8.9 88.6 1u4.8 94.2
vanillic aciu* 87.8 94.2 99.7 88.S
Caffeic aciu 1u8.9 112.S 1uS.S 11S.u
Syiingic aciu* 99.S 1u1.9 89.S 9S.6
Catechin* 9S.6 11u.7 11S.u 1uS.S
vanillin* 97.8 1uS.4 1u1.6 92.7
p-coumaiic aciu 96.8 1uS.8 1u1.1 8S
Syiingaluehyue* 8S.8 1uS.S 94.9 68.u
Epicatechin* 98.u 1u8.4 76.u 89.2
Feiulic aciu 9u.S 9S.6 86.u 1u2.9
Rutin 68.u 114.2 99.2 1uu.S
tians-iesveiatiol 9S.6 1uS.7 9S.7 96.u
Queicetin 116.9 1u8.8 1u9.7 11S.1
Kaempfeiol 88.u 1uu.2 88.1 88.6
*measuieu using LC-FL methou
















Capitulo 2



88
References

|1j Biavo, N. N.; Silva, S.; Coelho, A. v.; vilas-Boas, L.; Bionze, N.
R.; Anal. Chim. Acta 2uu6, 563, 84-92.
|2j Loieuana-La Toiie, u.; Saitta, N.; vilasi, F.; Pellicano, T.; Bugo,
u.; Food Chem. 2uu6, 94, 64u-6Su.
|Sj Robbins, R. }.; Bean, S. R.; J. Chromatogr., A 2uu4, 1038, 97-
1uS.
|4j Feinánuez-Pachon, N. S.; villaño, B.; Tioncoso, A. N.; uaicia-
Paiiilla, N. C.; Anal. Chim. Acta 2uu6, 563, 1u1-1u8.
|Sj Pioestos, C.; Bakogiannis, A.; Psaiianos, C.; Koutinas, A. A.;
Kanellaki, N.; Komaitis, N.; Food Control 2uuS, 16, S19-S2S.
|6j Slaukovský, R.; Solich, P.; 0ibánek, N.; J. Chromatogr., A 2uu4,
1040, 179-184.
|7j Keiem, Z.; Biavuo, B. A.; Shoseyov, 0.; Tugenuhaft, Y.; J.
Chromatogr., A 2uu4, 1052, 211-21S.
|8j Rouiiguez-Belgauo, N. A.; uonzález-Beinánuez, u.; Conue-
uonzález, }. E.; Péiez-Tiujillo, }. P.; Food Chem. 2uu2, S2S-
SS2.
|9j Bo, P.; Bogg, T. A.; Silva, N. C. N.; Food Chem. 1999, 64, 11S-
122.
|1uj Beinánuez, T.; Estiella, I.; Bueñas, N.; Feinánuez-ue Simon,
B.; Cauahia, E.; Eur. Food Res. Technol. 2uu7, 224, 69S-7uS.
|11j Caieii, N.; Coiiauini, C.; Elviii, L.; Nicoletti, I.; Zagnoni, I.; J.
Agric. Food Chem. 2uuS, 51, S226-S2S1.
|12j Rouiiguez-Belgauo, N. A.; Nalovaná, S.; Péiez, }. P.; Boiges,
T.; uaicia-Nontelongo, F.; J. Chromatogr., A 2uu1, 912, 249-
2S7.
|1Sj Bel Alamo-Sanza, N.; Nevaies-Bominguez, I.; Cáicel-Cáicel, L.
N.; Navas-uiacia, L.; Anal. Chim. Acta 2uu4, 513, 229-2S7.
|14j Bel Alamo, N.; Casauo, L.; Beinánuez, v.; }iménez, }. }.; J.
Chromatogr., A 2uu4, 1049, 97-1uS.
|1Sj Silva, L. R.; Anuiaue, P. B.; valentao, P.; Seabia, R. N.; Tiujillo,
N. E.; velásquez, E.; Food Chem. 2uuS, 89, 18S-189.
|16j Castellaii, N.; Saitini, E.; Fabiani, A.; Aifelli, u.; Amati, A.; J.
Chromatogr., A 2uu2, 973, 221-227.

Innovaciones en la ueteiminacion ciomatogiáfica ue antioxiuantes en alimentos



89
|17j Abeit-vian, N.; Tomao, v.; uallet, S.; Coulomb, P. 0.; Lacombe,
}. N.; J. Chromatogr., A 2uuS, 1085, 224-229.
|18j Rouiiguez-Biaz, R. C.; Aguilai-Caballos, N. P.; uomez-Bens,
A.; J. Sep. Sci. 2uu6, 29, 2772-2779.
|19j Suáiez, B.; Palacios, N.; Fiaga, N.; Rouiiguez, R.; J.
Chromatogr., A 2uuS, 1066, 1uS-11u.
|2uj Long, u. L.; Winefoiunei, }. B.; Anal. Chem. 198S, 55, 712A-
724A.
|21j uomez-Bens, A.; Aguilai-Caballos, N. P.; Trends Anal. Chem.
2uu2, 21, 1S1-141.
|22j Rouiiguez-Biaz, R. C.; Feinánuez-Romeio, }. N.; Aguilai-
Caballos, N. P.; uomez-Bens, A.; J. Agric. Food Chem. 2uu6, 54,
967u-9676.
|2Sj Rouiiguez-Biaz, R. C.; Feinánuez-Romeio, }. N.; Aguilai-
Caballos, N. P.; uomez-Bens, A.; Anal. Chim. Acta 2uu6, 578,
22u-226.
|24j Rieutoiu, A.; Piognon, P.; Biion, F.; Nahuziei, u.; Analyst
1997, 122, S9R-66R.




















Capitulo 2



9u

Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

91

Foou Chemistiy 124 (2u11) 17SS-17S9

Usefulness of terbium-sensitized luminescence
detection for the chemometric classification of wines
by their content in phenolic compounds

A. Andreu-Navarro, P. Russo, M.P. Aguilar-Caballos, J.M. Fernández-
Romero and A. Gómez-Hens (*)
Bepaitment of Analytical Chemistiy. "Naiie Cuiie Annex" Builuing, Campus
of Rabanales, 0niveisity of Coiuoba, E-14u71 Coiuoba, Spain


A methou foi wine classification baseu on the phenolic compounu
content, wine vaiiety anu geogiaphical aiea is uesciibeu. The methou
involves the use of the iesults obtaineu fiom the analysis of fifteen samples
of Italian anu Spanish wines fiom uiffeient geogiaphical oiigins |Sicilia
(Italy) anu Coiuoba (Spain)j using liquiu chiomatogiaphy (LC) with
photometiic anu fluoiimetiic uetection, in which eighteen phenolics weie
ueteimineu: gallic aciu, piotocatechuic aciu, p-hyuioxybenzoic aciu,
salicylic aciu, vanillic aciu, caffeic aciu, syiingic aciu, catechin, vanillin, p-
coumaiic aciu, syiingaluehyue, epicatechin, feiulic aciu, iutin, tians- anu
cis-iesveiatiol, queicetin anu kaempfeiol. Photometiic measuiements
weie peifoimeu selecting foui wavelengths (2S6, 28u, S2u anu S6S nm),
using a uioue aiiay uetection system. The fluoiimetiic uetection was
achieveu by measuiing the sensitizeu luminescence pioviueu by the
chelates foimeu between each analyte anu teibium (III). All samples weie
commeicial wines bought in local maikets anu analyzeu immeuiately aftei
they weie openeu. The pattein uata matiix was constiucteu by the
concentiation of each analyte piesent in wine, which was ueteimineu by
the most auequate methou, namely LC-photometiic oi LC-fluoiimetiic
methou. This uata matiix was subjecteu to uiffeient algoiithms in oiuei to
classify anu chaiacteiize the wine samples auequately. Supeiviseu (LBA)
Capitulo 2

92
anu un-supeiviseu (FA) pattein iecognition methous weie useu. The wine
pattein geneiation with LC sepaiation anu uual uetection appioach to
ueteimine eighteen phenolic compounus anu the chemometiic tieatment
pioviue an appiopiiate way with iecognition anu pieuiction iates. The
values obtaineu foi these iates weie 1uu % when fluoiimetiic uetection
was useu. These iesults can be consiueieu satisfactoiy, which pioves the
usefulness of the selecteu vaiiables.
Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

93
Introduction

In iecent yeais, theie has been an incieasing inteiest in the
uevelopment of chemometiic applications in oiuei to establish the vaiiety,
geogiaphical oiigin, manipulation anu othei technological featuies, which
uefine the taste of foous anu beveiages fiom agiicultuial oiigin |1-4j. The
ieasons foi this inteiest aie: 1) laws enfoice labeling of the geogiaphical
oiigin of the fooustuff anu beveiages in many countiies uue to uemanus of
moie infoimation fiom consumeis anu impiovement of theii uomestic
piouuction; 2) piouuceis have begun to auveitise theii bianus of high-
quality piouucts by incluuing geogiaphical oiigin anu othei featuies in the
label foi economic ieasons; S) pievention of fiauus anu auulteiation on the
label, piouuction anu commeicialization. Thus, the uevelopment of
methous giving acceptable infoimation is highly uesiiable foi consumeis,
piouuceis anu auministiative authoiities.

Wine inuustiy anu the maiket sectoi aie paiticulai examples in
which the uevelopment of sophisticateu chemometiic techniques aie
essential foi the impiovement of the analytical infoimation about wine
composition anu foi assessing wine authenticity |S,6j. Seveial chemometiic
pioceuuies have been useu as the basis foi uisciimination of wines
accoiuing to vinification technology anu classification accoiuing to iegion,
type anu vaiiety. Biffeient pattein iecognition techniques (PRT), such as
Piincipal Component Analysis (PCA) |7,8j, Lineai anu Canonical
Bisciiminant Analysis (LBA anu CBA) |9-11j, Piobabilistic Neuial Netwoik
(PNN) |12j, K-Neaiest Neighbois (KNN)|1Sj, Clustei Analysis (CA) |11j,
Nultiiegiession Analysis (NRA), Paitial Least Squaies (PLS) |14-16j, anu
CAINAN |17j have been useu foi this puipose. Theie is a gieat numbei of
combineu techniques employing gioup classification to evaluate wine
authenticity using uiffeient analytes such as volatile compounus |17j,
mineials anu tiace elements |14,18j aminoacius |14j anu phenolics |7,16j.

Phenolics aie impoitant constituents of wines which contiibute,
eithei uiiectly oi inuiiectly, to wine sensoiy piopeities (coloui,
astiingency, bitteiness anu aioma). Nany of these compounus have shown
potent antioxiuant activity, acting as bioactive agents that may contiibute
to pievent uegeneiative piocesses in human health |19j such as canceis,
caiuiovasculai uisoiueis, anu osteopoiosis, which aie associateu with
Capitulo 2

94
oxiuative uamage of BNA, pioteins anu lipius. The antioxiuants existing in
wines can help to pievent these uamages by ieacting uiiectly oi by
stimulating enuogenous piotection systems |2uj.

Phenolic compounus have been successfully useu foi assessing
wine authenticity anu classification pioceuuies since these compounus aie
chaiacteiistic of wine type anu can pioviue infoimation about the
geogiaphical oiigin. It is well known that uuiing feimentation the must in
contact with oxygen can unueigo oxiuation of phenolics, which causes wine
biowning. When the matuiation is finisheu, phenolic oxiuation uecieases
anu the phenolic content is stabilizeu. Liquiu chiomatogiaphy (LC) with
uioue-aiiay uetection (BAB) has been applieu to ueteimine the phenolic
fiaction |1Sj, which has been useu foi an effective wine, vinegai anu liquoi
classification anu pieuiction of the sample membeiship accoiuing to
employeu tieatment methous, vaiieties oi geogiaphical oiigin.

The aim of this papei has been the uevelopment of a methou foi
wine classification using the infoimation pioviueu by an LC methou with
photometiic anu fluoiimetiic uetection foi the ueteimination of eighteen
phenolic compounus |21j. These compounus aie gallic aciu (uAL),
piotocatechuic aciu (PTC), p-hyuioxybenzoic aciu (PBB), salicylic aciu
(SAL), vanillic aciu (vAI), caffeic aciu (CAF), syiingic aciu (SIR), catechin
(CAT), vanillin (vAI), p-coumaiic aciu (PC0), syiingaluehyue (SIA),
epicatechin (EPI), feiulic aciu (FER), iutin (R0T), tians- anu cis-iesveiatiol
(t-RSv anu c-RSv), queicetin (Q0E) anu kaempfeiol (KAE). The infoimation
about phenolic content of fifteen wine samples togethei with the vaiiety
(ieu, uiy sheiiy, iosé anu white), anu geogiaphical aieas of these wine
samples, have been useu foi classification puiposes in this stuuy. The
photometiic uetection was accomplisheu at foui wavelengths, wheieas the
fluoiimetiic uetection involveu the post-column ueiivatization of ten of
these compounus with teibium(III) to foim the coiiesponuing luminescent
chelates. The geogiaphical aieas have been also sepaiateu in foui types of
wines accoiuing to the local oiigin: Coiuoba anu Nontilla-Noiiles (Spain)
anu Sicilian anu Naisala (Italy) wines. An auuitional objective of this papei
has been to eluciuate which methou (LC-photometiic oi LC-fluoiimetiic)
pioviues the best pathway to classify wines accoiuing to theii vaiiety anu
geogiaphical oiigin. Although only fifteen wine samples have been useu to
uevelop the pioposeu methou, a similai numbei of samples, in the iange of
Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

95
9-22 samples, have been useu in othei chemometiic stuuies with
satisfactoiy iesults |7,16j.

1. Experimental

1.1. Wine samples

The fifteen samples of uiffeient Italian anu Spanish wines weie
bought fiom local maikets in sealeu 7Su-ml bottles. The samples analyseu
weie ieu (WR, five samples), iosé (WS, one sample), "uiy sheiiy" (W0, foui
samples) anu white (WW, five samples) wine samples. Samples WRuu1,
WRuuS, WRuu4 anu WSuu1 weie fiom Coiuoba, samples WRuu2, WRuuS
WWuu2 anu WWuuS weie fiom Sicilia, samples W0uu1, W0uu2 anu
W0uuS weie fiom Naisala , anu samples W0uu4, WWuu1, WWuu4 anu
WWuuS weie fiom Nontilla-Noiiles. Each sample was uiluteu with
uistilleu watei to match uynamic calibiation ianges anu then, uiiectly
injecteu onto the chiomatogiaphic system anu tieateu following the
pioceuuie below inuicateu. Each ueteimination was assayeu in tiiplicate.

1.2. LC-separation and photometric/fluorimetric detection

An Agilent 12uu seiies liquiu chiomatogiaphy system (Agilent
Technologies España, Las Rozas, Spain) composeu by a quateinaiy pump, a
uegassei unit, a vial autosamplei, a theimostateu column compaitment anu
a uioue aiiay uetectoi, was useu. An SLN Aminco (SLN Instiuments,
0ibana, IL, 0SA) AB2 luminescence spectiometei pioviueu with a 1Su W
continuous xenon lamp anu a 7 W pulseu xenon lamp, fuinisheu with a
176-uS2-QS Bellma (Bellma Bispania, Baicelona, Spain) flow cell with an
innei volume of 18 ml was useu foi fluoiescence measuiements. A uilson
(uilson Inc., villieis-le-Bel, Fiance) Ninipuls S low-piessuie peiistaltic
pump anu 0mnifit (Biba Inuustiies Ltu., Cambiiuge,0K) Teflon tubing of
u.S mm i.u. weie also useu foi constiucting the post-column ueiivatization
manifolu. Chiomatogiaphic sepaiation was peifoimeu using an 0nyx
monolithic C
18
column (Phenomenex,Toiiance, 0SA) 1uu mm x 4.6 mm i.u.,
poie sizes: mesopoies (1S nm), maciopoies (2 nm). Stanuaius oi uiluteu
samples (Su ml), containing the analytes at concentiations within theii
coiiesponuing uynamic ianges, weie injecteu into the column. Betails
conceining ieagent anu solutions, elution piogiamme, photometiic
Capitulo 2

96
uetection, on-line ueiivatization anu fluoiimetiic uetection aie uesciibeu
elsewheie |21j. The time necessaiy to achieve LC-photometiic anu LC-
fluoiimetiic chiomatogiams was 2S min. Photometiic uetection was
accomplisheu at 2S6 nm (foi PTC, PBB, vAI, R0T), 28u nm (foi uAL, SAL,
SIR, CAT, vAA, PC0, EPI, c-RSv), S24 nm (foi CAF, SIA, FER, t-RSv) anu S6S
nm (foi Q0E, KAE), which coiiesponu to the maximum absoiption
wavelength of each phenolic compounu. Aftei each injection, a column
cleaning-up anu conuitioning step was intiouuceu to have the
chiomatogiaphic system ieauy foi the next opeiation aftei S min. The
fluoiimetiic ueiivatization ieagent was teibium(III), which foims
fluoiescent chelates with the following analytes: uAL, PTC, PBB, SAL, vAI,
SIR, CAT, vAA, SIA anu EPI. The fluoiescence was monitoieu at i
ex
S17, i
em

S4S nm foi 2u min, anu the coiiesponuing blank solutions weie subtiacteu.
Chiomatogiams weie taken using the oiiginal softwaie of the luminescence
spectiometei anu the iaw uata of luminescence intensity anu time weie
expoiteu anu tieateu using auequate softwaie packages foi the estimation
of the main chiomatogiaphic paiameteis.


2. Results and discussion

2.1. Analytical features

Calibiation giaphs weie iun unuei the optimum expeiimental
conuitions by using an exteinal calibiation methou. Table 1 shows the
ietention time foi each phenolic, the calibiation paiameteis anu the
uetection limits (L0Bs) obtaineu foi both methous, which weie calculateu
accoiuing I0PAC iecommenuations |22j.


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Innovaciones en la determición cromatográfica de antioxidantes en alimentos

99
2.2. Statistical and Preliminary data exploration

All the statistical analyses weie peifoimeu by means of the
statistical softwaie package STATuRAPBICS Plus (Statgiaphics Consulting,
Softwaie Cientifico S.L., Nauiiu, Spain) foi Winuows S.1 anu SPSS 1S.u
(SPSS Inc., Chicago, IL, 0SA) foi Winuows S.1 using a Bewlett Packaiu
BP418uC (Bewlett Packaiu España, Las Rozas, Spain) as giaphic output. LC
analysis was peifoimeu in tiiplicate anu the uata aie given as inuiviuual
values ± stanuaiu ueviation (S.B.). Each analyte was piocesseu accoiuing
with the selecteu sepaiation¡ueiivatization anu uetection moue. Table 2
summaiizes the phenolic content of each wine sample, which was obtaineu
fiom the chiomatogiaphic methous uepicteu in Table 1. This infoimation
was auueu to a matiix of uata, which also incluueu the uata coiiesponuing
to wine vaiiety (Wv) anu geogiaphic oiigin (u0). The mean values
obtaineu in the uiffeient gioups weie compaieu by 0ne-way AN0vA anu
the t-test, assuming that theie weie significant uiffeiences between mean
values when statistical compaiison gave p<u.uS. In multivaiiate analysis
each sample was consiueieu to be a uata vectoi iepiesenteu by the
concentiation of the selecteu phenolic compounus. Nissing obseivations
can be pioblematic in multivaiiate analysis, anu sometimes measuiements
cannot be computeu if theie aie missing values in the seiies. 0sually, these
missing values appeaieu when a paiticulai obseivation was un-known oi
when the concentiation value was lowei than the uetection limit. Bence,
thiee uiffeient alteinatives foi ieplacing missing values weie assayeu: the
ieplacement by the seiies mean (NEA), by half of the value of L0B (L0B¡2)
anu by half of the value of L0Q (L0Q¡2).


Table 2. Phenolic content in the analyzed samples
Compounu
a

Sample coue
WRuu1 WRuu2 WRuuS WRuu4 WRuuS WSuu1 W0uu1 W0uu2
uallic aciu* 2.8 ± u.1 98.u ± 4.u Su.S ± u.8 SS± 1 87.2± u.8 S.6u ± u.S 9.4S ± u.u7 1u.9 ± u.2
Piotocatechuic aciu* 1.268 ± u.uu2 1.6 ± u.1 1.u9 ± u.u1 1.269 ± u.uu1 2.2 ± u.2 u.S8 ± u.u1 1.61S ± u.uu1 1.Su ± u.u8u
p-hyuioxy benzoic aciu* u.8 ± u.u1 n.q. u.26S ± u.uuS n.u. n.q.. u.6S ± u.u2 u.S44 ± u.uu4 u.48 ± u.u4
Salicylic aciu* n.q. n.u. n.q. u.S2± u.u2 n.u. n.u. u.2u ± u.u1 u.886 ± u.uu2
vanillic aciu* 1.21u ± u.u2 1.2S ± u.uS 1.u2u ± u.uuS u.61u ± u.uuS 2.2 ± u.1 u.SS ± u.u6 u.27 ± u.u1 u.S4 ± u.u2
Caffeic aciu 2.9u ± u.u4 n.q. 9.7 ± u.1 1u.7 ± u.S 7.27 ± u.u7 1.71 ± u.u2 1.81 ± u.u1 1.S74 ± u.uu6
Syiingic aciu* 4.18 ± u.u2 6.S± u.4 S.46 ± u.uS u.S7 ± u.uS S.1 ± u.S 1.4 ± u.1 u.2uu ± u.uu8 u.46 ± u.u1
Catechin* 26.S ± u.2 S.8± u.2 Su ± 2 8.1 ± u.S SS ± 2 n.u. n.u. 1.uu ± u.u2
vanillin* n.q. n.u. n.u. n.u. n.u. n.q. n.u. n.q.
p-coumaiic aciu 6.4 ± u.1 16.S ± u.2 11.8 ± u.2 11.6 ± u.S 24.6 ± u.2 u.11 ± u.u1 2.u4 ± u.uS 1.296 ± u.uu6
Syiingaluehiue* 1.S ± u.1 S.u ± u.2 1.6 ± u.1 n.u. 2.12 ± u.uS n.u. n.q. n.u.
Epicatechin* 14.u ± u.1 S2.u ± u.S 1S.S ± u.S 29.1 ± u.1 26.S ± u.S 1.94 ± u.8u u.SS2 ± u.uu6 u.S2 ± u.u2
Feiulic aciu u.S99 ± u.uu4 u.46 ± u.u2 u.44S ± u.uuS u.S6 ± u.u2 u.S6 ± u.u1 u.14 ± u.u2 1.uu ± u.uS u.S4 ± u.u2
Rutin n.u. 19.u ± 1.u S.SS ± u.u2 u.7S ± u.uS S.S ± u.2 u.81 ± u.u2 u.961 ± u.uu1 u.46 ± u.u2
tians-iesveiatiol u.42 ± u.u2 u.S4 ± u.u1. u.46u ±u.uu4 u.u94 ± u.uu2 u.72 ± u.uS u.8S ± u.uS n.u. u.uSu ± u.uu2
Queicetin S.S ± u.2 11.24 ± u.uS 6.S ± u.2 7.u ± u.S 1S.u ± u.S u.47 ± u.u2 n.u. n.u.
Kaempfeiol u.S2 ± u.u1 u.216 ± u.uu2 u.S6u ± u.uu9 u.49u ± u.uuS u.47 ± u.u1 n.q. n.u. n.u.
*measuieu using LC-FL methou,
a
Content in mg L
-1
, Nean ± SB (n = S), (n.u.: below uetection limit; n.q.: not quantifieu, below
quantification limit











Table 2. Continuation.
Compounu
a

Sample coue
W0uuS W0uu4 WWuu1 WWuu2 WWuuS WWuu4 WWuuS
uallic aciu* 1S.S ± u.9 1.7u ± u.uS 1.9 ± u.1 2.u ± u.1 2.6 ± u.1 2.16 ± u.u8 1.84 ± u.u8
Piotocatechuic aciu* 1.642 ± u.uuS u.77 ± u.u2 1.14 ± u.u6 u.6S ± u.u1 u.41 ± u.u4 1.S24 ± u.uu4 u.868 ± u.uu2
p-hyuioxy benzoic aciu* u.17S ± u.uuS n.q. n.q. u.2S2 ± u.uu4 u.11 ± u.u4 u.S4± u.u4 u.79 ± u.u2
Salicylic aciu* u.S2 ± u.u2 u.SS ± u.u2 u.21S ± u.uu1 u.u9u ± u.uu4 n.q. u.21 ± u.u1 u.24u ± u.uu8
vanillic aciu* u.29 ± u.u2 u.Su ± u.uS 1.26 ± u.u4 u.114 ± u.uu8 u.1u6 ± u.uu8 1.16 ± u.u8 u.2u8 ± u.uu8
Caffeic aciu S.2u ± u.u1 u.674 ± u.uuS u.16S ± u.uuS 1.S12 ± u.uu6 1.4S ± u.u1 n.q. 1.68 ± u.u8
Syiingic aciu* u.1S9 ± u.uu2 u.Su ± u.u2 u.S6 ± u.uS u.Su ± u.u1 u.128 ± u.uu4 u.8u ± u.uS u.u9u ± u.uuS
Catechin* n.u. n.u. u.9S ± u.uS S.2 ± u.1 1.Su ± u.u6 1.2 ± u.1 n.u.
vanillin* n.q. n.u. n.u. n.u. u.2u ± u.u2 u.S6 ± u.u4 n.u.
p-coumaiic aciu 1.68 ± u.uS n.u. u.S1 ± u.u1 u.S9u ± u.uu4 u.6u ± u.u4 n.u. u.91 ± u.uS
Syiingaluehiue* n.u. u.4S ± u.uS n.u. n.q. u.14 ± u.u1 u.4S ± u.u2 n.u.
Epicatechin* u.Su ± u.u4 n.u. n.q. 1.S2 ± u.1 u.4S ± u.u2 2.9 ± u.S n.u.
Feiulic aciu u.S4u ± u.uuS u.42 ± u.u4 n.q. u.486 ± u.uu6 u.S44 ± u.uu2 u.21 ± u.u2 u.47 ± u.u2
Rutin u.6S ± u.uS n.q. n.u. u.7S ± u.u2 u.2u ± u.u2 u.212 ± u.uu4 n.q.
tians-iesveiatiol n.q. n.q. u.u1S ± u.uu7 u.u4u ± u.uu4 u.u84 ± u.uu2 n.q. n.q.
Queicetin n.u. u.S9 ± u.u1 n.u. n.u. n.u n.u. u.S2S ± u.uuS
Kaempfeiol n.u. n.q. n.u. n.u. n.u. n.u. n.u.
*measuieu using LC-FL methou,
a
Content in mg L
-1
, Nean ± SB (n = S), (n.u.: below uetection limit; n.q.: not quantifieu, below
quantification limit


Capitulo 2

102
A seconu tieatment, consisting on the noimalization of the uata in
oiuei to avoiu initial weighing influences on the concentiation values piioi
to the classification analysis, was also assayeu. Two noimalization
pioceuuies weie testeu: inteival scaling (IS) anu autoscaling (AS). These
uiffeient stiategies leau to the selection of the methou, pioviuing the best
sample classification. Finally, the selecteu uata matiices weie constiucteu
using IS anu L0Q¡2 pie-piocessing methous as noimalizeu anu hole
ieplacing pioceuuies |1S,2Sj. Thiee uata matiices weie constiucteu using
the following stiuctuie: a fiist column incluuing wine vaiiety, a seconu
column incluuing the geogiaphical iegion anu uiffeient columns incluuing
the concentiation of the selecteu analytes. The matiix type A coiiesponus
to the concentiation of seventeen analytes (uAL, PTC, PBB, SAL, vAI, PC0,
SIR, CAF, vAA, SIA, CAT, EPI, FER, R0T, Q0E, KAE anu t-RSv) using LC-
photometiic anu LC-fluoiimetiic appioaches. The analyte c-RSv pioviueu
incomplete infoimation foi the selecteu fifteen wines anu, thus, it was
iemoveu. The seconu matiix, namely type B, was geneiateu using the
infoimation pioviueu by ten analytes quantifieu by LC-fluoiimetiic
appioach (uAL, PTC, PBB, SAL, vAI, SIR, CAT, vAA, SIA anu EPI). Finally, the
matiix type C was constiucteu using the infoimation pioviueu by sixteen
analytes quantifieu by the LC-photometiic methou (uAL, PTC, PBB, vAI,
CAF, SIR, CAT, vAA, PC0, SIA, EPI, FER, R0T, t-RSv, Q0E anu KAE). Paitial
infoimation pioviueu by SAL anu c-RSv weie not useu foi ueveloping this
matiix.

2.3. Factor Analysis using Principal Components

Factoi Analysis (FA), using piincipal components as the methou foi
factoi extiaction, was employeu to summaiize the infoimation in a ieuuceu
factoi numbei. This pioceuuie allows ieuucing uata uimensionality, a uata
exploiation that investigates how many components (a lineai combination
of oiiginal vaiiables) aie necessaiy to explain the moie significant paits of
the vaiiance in the methou with a minimum loss of infoimation. Table 3
summaiizes the featuies of the FA achieveu using the thiee uata sets,
namely A, B anu C, which incluueu the initial Eigenvalues foi the piincipal
component founu, theii peicentage of vaiiance anu the Cummulative
peicentage of vaiiance. This Table also uepicts the vaiiable influence
obtaineu foi each PCA aftei vaiimax Rotation incluuing theii Communality
values. The fiist type of FA (type A uata) was peifoimeu using the analyte
Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

103
concentiations obtaineu using both LC-photometiic anu LC-fluoiimetiic
infoimation. The objective of this FA is to obtain a ieuuceu numbei of
factois which explain the vaiiability of the selecteu analytes. When this
uata set was useu, the fiist five factois weie chosen (87.6S % of the total
vaiiance) because the eigenvalues weie highei than 1, anu theiefoie, they
explain moie vaiiance than each oiiginal vaiiable. All vaiiables piesent a
communality equal to oi highei than u.649, anu theiefoie, they aie well
iepiesenteu by the five factois. A vaiimax iotation was caiiieu out to
minimize the numbei of vaiiables that influence each factoi, anu then, to
facilitate the inteipietation of the iesults. The fiist factoi that explains the
highei peicentage of vaiiance (S2.u4 %) is mainly associateu with SIA, SIR,
R0T, uAL anu Q0E. The seconu factoi that explains the 11.uu % of the total
vaiiance is ielateu to CAF anu KAE anu CAT. The iemaining factois
selecteu, with 24.6u % of the total vaiiance explaineu, aie ielateu to FER
(thiiu factoi) anu PBB (fifth factoi). 0n the basis of the fiist FA, these 1u
vaiiables can be useu to iepeat the FA obtaining similai iesults with
iespect of the oiiginal FA anu, thus, woulu be selecteu in fuithei
chemometiic analysis without appieciable vaiiation.

The seconu FA stuuy incluuing vaiimax iotation was peifoimeu
using the infoimation of the ten analytes ueteimineu by the LC-fluoiimetiic
methou (type B). Foui factois weie extiacteu fiom this FA with eigenvalues
highei than 1, which explains the 87.Su % of the vaiiability fiom the
oiiginal vaiiables (see Table 3). The fiist factoi that explains the highei
peicentage of vaiiance (S2.S7 %) is mainly associateu with SIR, SIA, vAI,
uAL anu EPI. The othei thiee factois explain the SS.17 % of the whole
vaiiability anu weie associateu with PBB (seconu factoi), SAL (thiiu factoi)
anu vAA (fouith factoi).










Table 3. Features of Factor Analysis
a

Bata set
Initial Eigenvalues
vaiiables aftei vaiimax Rotation (Communality values)
b

Factoi
Numbei
Total
% of
vaiiance
Cumulative
%
Type A
17 analytes
(1u ieuuceu
vaiiables)
(photometiic anu
fluoiimetiic)
1 8.847 S2.uS8 S2.uS8 SIA (u.9S2), SIR (u.9u7), R0T (u.9uS), uAL (u.897)
2 1.871 11.uu8 6S.u46 CAF (u.9S2), KAE (u.897), CAT (u.816)
S 1.8u2 1u.S99 7S.64S FER (u.769)
4 1.S42 7.89S 81.SS8 -
S 1.uS9 6.119 87.647 PBB (u.894)

Type B
1u analytes
(8 ieuuceu
vaiiables)
(fluoiimetiic)
1 S.2S7 S2.S66 S2.S66 SIR (u.9S7), SIA (u.898), vAI (u.879), uAL (u.8u8), EPI (u.8u4)
2 1.S41 1S.41u 6S.776 PBB (-u.944)
S 1.119 11.188 76.964 SAL (u.86u)
4 1.uSS 1u.SSS 87.296 vAA (u.97u)

Type C
16 analytes
(1u ieuuceu
vaiiables)
(photometiic)
1 7.117 44.48S 44.48S SIA (u.94S), vAI (u.9u4), R0T (u.896), PTC (u.862), EPI (u.816)
2 S.6S6 22.72S 67.2u8 KAE (u.911), PC0 (u.892), CAF (u.861)
S 1.9S2 12.2uu 79.4u8 SIR (u.742)
4 1.uS9 6.49S 8S.9uS FER (-u.9uu)


a) Extraction method using Principal Component Analysis with Kaiser Normalization, b) using Principal Component Analysis
Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

105
The last FA stuuy was peifoimeu using infoimation fiom the
sixteen analytes quantifieu by the LC-photometiic methou (type C). As can
be seen in Table 3, foui factois weie extiacteu (8S.9u %), in which the fiist
factoi explains the 44.48 % of vaiiance with associateu vaiiables of SIA,
vAI, R0T, PTC anu EPI. The seconu factoi was associateu to the vaiiables
KAE, PC0 anu CAF with a vaiiance peicent of 22.7S %. Finally, SIR (thiiu
factoi) anu FER (fouith factoi) iepiesent the last 18.69 % of the vaiiability.
The uata sets A anu B shows high coinciuence in the iesults with at least
foui of the vaiiables at the same significance level (SIR, SIA, uAL anu PBB)
anu exhibits vaiiance peicentages quite similai. The FA type C also
pioviues a ieuuction of the vaiiables in an acceptable concoiuance level
with the FA type A (also foui vaiiables). Bowevei, othei six vaiiables weie
not concoiuant at the significance level, which hinueis theii applicability in
fuithei chemometiic analysis. As a iesult of Factoi Analysis, the new latent
vaiiables, which aie a lineai combination of the initial expeiimental
vaiiables, iepiesent a similai significance uegiee in the vaiiability of the
initial expeiimental vaiiables. These vaiiables weie also useu in fuithei
chemometiic tieatments.

2.4. Discriminant analysis

Lineai uisciiminant analysis (LBA) is a supeiviseu classification
pioceuuie in which the numbei of categoiies anu the samples that belong
to each categoiy aie pieviously uefineu. This statistical technique is baseu
on the extiaction of lineai uisciiminant functions of the inuepenuent
vaiiable by means of a quantitative uepenuent vaiiable (categoiy) anu the
quantitative inuepenuent vaiiables. Cuiiently, two uiffeient piocesses can
be applieu in LBA: 1) Stepwise LBA that selects the quantitative vaiiables
which enhanceu uisciimination of the gioups establisheu by the uepenuent
vaiiable. Foi this puipose, the ciiteiia foi this selection is the Wilks'
lambua, which is a measuiement of how well each function sepaiates
objects (samples) into gioups, anu it is equal to the iatio of the total
vaiiance in the uisciiminant scoies not explaineu by uiffeiences among the
gioups. Smallei values of Wilks' lambua inuicate gieatei uisciiminatoiy
ability of the function. The associateu chi-squaie statistic test hypothesizes
that the means of the function listeu aie equal acioss gioups anu the small
significance values inuicate that the uisciiminant function uoes bettei than
chance at sepaiating the gioups; 2) Intiouuction of all inuepenuent
Capitulo 2

106
vaiiables. The objective of this piocess is to keep all the oiiginal
infoimation, although the system obtaineu is moie complex. This appioach
is applieu when the stepwise methou cannot piouuce a goou classification
of the samples as a function of the quantitative vaiiable.

Accoiuing to the uata set, a LBA was uevelopeu, using new matiices
of uata integiateu by the ieuuceu oiiginal vaiiables, which weie selecteu
fiom to the pievious FA. Each of the ieuuceu uata set of types A, B anu C
was also subjecteu to the LBA, consiueiing two types of classification
ciiteiia: accoiuing to the wine vaiiety (ieu, uiy sheiiy, iosé anu white) oi
the geogiaphical oiigin (Coiuoba, Nontilla-Noiiles, Sicilia anu Naisala).
The iesults of the LBA weie valiuateu using the leave-on-out cioss-
valiuation. In this pioceuuie, a uefineu numbei of samples weie
sequentially iemoveu fiom the oiiginal uata set anu the classification
mouel was iebuilt. All the samples of the uata set aie sequentially iemoveu
anu classifieu. Finally, aftei a significant numbei of iteiations, the objects
weie classifieu anu the peicentage of coiiect classification is given in all
instances. The classification iesults accoiuing to the wine vaiiety anu
geogiaphical oiigin aie shown in Table 4. As can be seen, classification
paiameteis such as numbei of eigenvalues, lineai coiielation, Wilks'
lambua anu Chi-squaie coefficients ieveal acceptable aggiegation foi at
least thiee of the uisciiminant factois uefineu, in which the quantitative
values of the vaiiables coiielate well with the pievious FA. The pieuicteu
iesults pioviueu a peicentage of pieuicteu membeiship values accoiuing to
the wine vaiiety of 7S.S % using the LC-photometiic methou anu 97.92 %
foi the combination of LC with fluoiimetiic anu photometiic uetection. The
seconu attempt to classify the objects accoiuing to the geogiaphical oiigin
pioviueu veiy similai iesults, in which the uisciiminant paiameteis anu
the classification iesults weie achieveu using the ieuuceu uata sets A, B anu
C. The pieuiction membeiship exhibiteu pieuiction peicentages between
91.1u anu 97.8u %. Bowevei, when the cioss-valiuation pioceuuie is useu,
these values ueciease to 86.7 % anu 9S.6 %.





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Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

111
Fiom the iesults obtaineu by the classification stuuies, accoiuing to
the vaiiety of wines oi to theii geogiaphical oiigin, it can be concluueu that
the best sepaiation anu, thus, classification is obtaineu using the
infoimation pioviueu by the LC-fluoiimetiic methouology (uata set B). As
can be seen, this uata matiix only pioviues a paitial ieuuction in two
vaiiables when compaieu to the oiiginal type B matiix (pieuiction of 9S.Su
%). If the Lineai Bisciiminant Analysis using the oiiginal uata set (1u
vaiiables achieveu using the LC-fluoiimetiic uetection pioceuuie) is
uevelopeu, a significant impiovement of the peicentage of pieuicteu
sample membeiship using wine vaiiety oi geogiaphical oiigin as
classification factoi was achieveu. Table 5 shows the classification
paiameteis anu the peicentage of the pieuicteu membeiship obtaineu. As
can be seen, the peicentage of pieuiction was complete in all instances
(1uu % of the pieuicteu membeiship). Figure 1 uepicts the classification of
wines accoiuing to the vaiiety (A) anu geogiaphical oiigin (B). As can be
seen, the similaiities between wines of the same vaiiety oi the same
geogiaphical oiigin aie well establisheu in all instances, leauing to a coiiect
uisciimination among the uiffeient gioups.












Figure 1. Pieuiction examples using LBA foi vaiieties (a) anu geogiaphical oiigins (b) of the
wines using ieuuceu uata matiix type B (n=1u) foi LC-fluoiimetiic methou.
Symbol foi wine classification accoiuing vaiiety, ( ) uenotes ieu, ( ) uiy sheiiy,
( ) iosé anu ( ) white, iespectively, anu foi geogiaphical oiigin, ( ) uenotes
Coiuoba (Spain), ( ) Nontilla-Noiiles, ( ) Sicilia (Italy) anu ( ) Naisala.

Capitulo 2

112
3. Conclusions

Accoiuing to the iesults obtaineu, it shoulu be emphasizeu that: a)
It has been establisheu that the LC-fluoiimetiic uetection methou using a
combineu technique of FA¡LBA pioviues an auequate pathway to classify
the wines accoiuing to theii vaiiety oi geogiaphical oiigin, b) The
pioposeu methou constitutes an oiiginal pioceuuie to classify wines using
LC anu optical uetection, c) The fluoiescence of the ueiivatizeu analytes
pioviues a quantitative anu selective methou, using a ieuuceu numbei of
analytes, to uevelop an auequate chemometiic classification pioceuuie, anu
u) A coiiect classification pathway with 1uu % of the coiiect pieuicteu
membeiship is achieveu by selecting only a ieuuceu numbei of phenolic
compounus, such as hyuioxybenzoic acius (uAL, PTC, PBB, SAL, vAI anu
SIR), phenolic aluehyues (SIA anu vAA) anu flavonoius (R0T anu KAE).

Acknowledgement

Authois giatefully acknowleuge the finantial suppoit to the Anualusian
Autonomous uoveinment uiant (uiant FQN 1SS6). P.R. is also giateful to
Eiasmus Fellowship Piogiam.
Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

113
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199S, 69, 1SS-14u.
|4j Smith, R.u.; }. Agiic. Foou Chem. 2uuS, SS, 4u41-4u4S.
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Kallithiaka, S.; Tienus Foou Sci. Technol.1999, 1u, S21-SS6.
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}.P.; }. Agiic. Foou Chem. 2uuS, S1, 4SuS-4Su7.
|1Sj Beltián, N.B.; Buaite-Neimouu, N.A.; Bustos, N.A.; Salah, S.A.;
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|14j Capion, X.; Smeyeis-veibeke, }.; Nassait. B.L; Foou Chem. 2uu7,
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|1Sj Nassait, B.L; Banubook of Chemometiic anu Qualimetiies,
Elseviei Sciences. Amsteiuam, 1998.
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}ouijon, F. ; Anal. Chim. Acta. 2uu8, 621, 8-18.
|17j Ballabio, B.; Nauii, A.; Toueschini, R. ; Buiatti, S. ; Anal. Chim. Acta.
2uu6, S7u, 249-2S8.
|18j Speikova }.; Suchanek, N.; Foou Chem. 2uuS, 9S, 6S9-66S.
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Capitulo 2

114
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Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

115


}. Sep. Sci. 2u1u, SS, Su9-S1S

Luminescent determination of flavonoids in orange
juices by liquid chromatography with post-column
derivatization with aluminium and terbium

A. Andreu-Navarro, J.M. Fernández-Romero, A. Gómez-Hens
(*)

Bepaitment of Analytical Chemistiy, Faculty of Sciences, 0niveisity
of Coiuoba, "Naiie Cuiie Annex" Builuing. Campus of Rabanales, E-
14u71 Coiuoba, Spain


A new post-column ueiivatization system is uesciibeu anu applieu
to the ueteimination of flavonoius in citiic beveiages aftei theii sepaiation
by liquiu chiomatogiaphy using a monolithic column. The ueiivatization
involves the foimation of the chelates of the analytes with aluminium (III)
anu teibium (III) in the piesence of the suifactant souium uouecylsulfate
anu the measuiement of the teibium sensitizeu luminescence at i
ex
S6u anu
i
em
S4S nm. Naiingin, hespeiiuin, queicetin, naiingenin, anu kaempfeiol
have been chosen as analyte mouels. The laige Stokes shift anu the
ielatively long wavelength emission of teibium(III) can minimize
inteifeiences fiom backgiounu sample matiix, which usually emit at
shoitei wavelengths. Calibiation giaphs weie constiucteu in the inteivals
6.u - 17uu ng¡mL naiingin, 9.8 - 17uu ng¡mL hespeiiuin, 2.1 - 2uuu ng¡mL
queicetin, S.2 - 1Suu ng¡mL naiingenin anu 2.S - 2uuu ng¡mLkaempfeiol,
with iegiession coefficients highei than u.99SS in all instances. The
piecision of the methou, expiesseu as RSB%, was establisheu at two
concentiation levels, with values of 1.S anu 4.7 %, which coiiesponueu to
the minimal anu maximal eiioi zones of the calibiation giaphs. The
piactical usefulness of the methou is uemonstiateu by the analysis of
oiange juices, which weie uiluteu anu uiiectly injecteu into the
chiomatogiaphic system, obtaining iecoveiies between 86.9 anu 1u8.2 %.
Capítulo 2

116
Introduction

Flavonoius aie a gioup of polyphenolic compounus wiuely
uistiibuteu in vegetables anu fiuits |1j. The ueteimination of these
compounus is of gieat inteiest owing to theii multiple biological effects,
incluuing antioxiuant activity, antitumoi, antimutagenic, antibacteiial anu
angiopiotective piopeities |2, Sj. They also contiibute to uiffeient plant
piopeities such as coloui, flavoui, fiagiance, nutiition, stability anu
theiapeutic piopeities. Flavonoius as 2-phenyl-benzo-α-pyiones, aie
classifieu accoiuing to the multituue of substitution patteins in the two
bencene iings of theii basic stiuctuie. vaiiation in theii heteiocyclic iings
gives iise to flavonols, flavones, catechins, flavanones, anthocyaniuins anu
isoflavones |4j. The flavonoiu content in oiange juices has special inteiest
as it contiibutes to the quality of these samples |Sj.

The main sepaiation technique applieu to the ueteimination of
flavonoius in oiange juices is liquiu chiomatogiaphy (LC) |6-11j, using
ieveiseu-phase columns, although gas chiomatogiaphy |12j, involving the
foimation of tiimethylsilyl ueiivatives, has been also uesciibeu foi this
puipose. The usefulness of a monolithic column, which can opeiate at
highei flow-iates with lowei back-piessuies than conventional columns,
has been pieviously uesciibeu foi the LC sepaiation of some flavonoius in
wine samples |1Sj. Bowevei, to the best of oui knowleuge, this type of
column has not been useu up to uate foi the ueteimination of these
compounus in oiange juices.

Photometiic |6-1uj, coulometiic |1uj anu mass spectiometiic (NS)
|6, 11j uetection systems have been iepoiteu in the LC methous uesciibeu
foi the analysis of these samples. Some flavonoius, such as queicetin anu
kaempfeiol, have been fluoiimetiically ueteimineu in bouy fluius using
aluminium(III) as post-column ueiivatization ieagent to obtain fluoiescent
chelates |14j. Anothei post-column ueiivatization system pieviously
uesciibeu foi the ueteimination of these compounus in wine samples
involves the foimation of the coiiesponuing teibium(III) chelates anu the
measuiement of the sensitizeu luminescence |1Sj. This phenomenon is an
inteimoleculai eneigy tiansfei piocess, in which the liganu acts as uonoi
anu teibium(III) acts as acceptoi anu emits luminescence, that allows veiy
Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

117
sensitive anu selective ueteiminations, although it use as uetection system
in LC has been ielatively limiteu |1Sj

This aiticle iepoits foi the fiist time the use of a monolithic column
foi the uiiect sepaiation of flavonoius in oiange juice samples anu a new
post-column ueiivatization system foi the luminescent uetection of these
compounus. The ueiivatization system is baseu on the capability of
flavonoius to foim stable chelates with aluminium(III) anu teibium(III) anu
the measuiement of the teibium sensitizeu luminescence at i
ex
S6u anu i
em

S4S nm, in the piesence of a sub-micellai meuium pioviueu by souium
uouecylsulfate (SBS). The laige Stokes shift anu the ielatively long
wavelength emission minimize potential inteifeiences fiom backgiounu
sample matiix, which usually emit at shoitei wavelengths. Two flavone
aglycones (queicetin anu kaempfeiol), a flavanoneaglycone (naiingenin)
anu two flavanone-0-glycosiues (hespeiiuin anu naiingin) (Figure 1) have
been chosen as mouel analytes, since they aie the most iepiesentative
flavonoius in oiange juices. The flow iate useu was lowei than that usually
ieacheu using a monolithic column, since it was necessaiy to make
compatible the sepaiation piocess with the post-column system. Bowevei,
the use of this column impioves the chiomatogiaphic iesolution anu the
sepaiation of the analytes iequiies a shoitei time than that using a
conventional column (7-9).

(A) (B)





Type of polyphenols Name
Abbieviati
on
Substituents
R1 R2 RS R4
Flavanone 0-glycosiue (A) Naiingin NAR B ulycosiue B 0B
Flavanone 0-glycosiue (A) Bespeiiuin BES B ulycosiue 0B 0CBS
Flavanoneaglycone (A) Naiingenin NAN B B B 0B
Flavone aglycone (B) Queicetin Q0E 0B B 0B 0B
Flavone aglycone (B) Kaempfeiol KAE 0B B B 0B

Figure 1.Chemical stiuctuies anu nomenclatuie of the analytes selecteu in this
ieseaich.
Capítulo 2

118
1. Experimental

1.1. Instrumentation

An Agilent 12uu Seiies Liquiu Chiomatogiaphy System composeu
by a quateinaiy pump, a uegassei unit, a vial autosamplei, a theimostateu
column compaitment has been useu. An SLN Aminco (0ibana, IL) AB2
luminescence spectiometei pioviue with a 1Su W continuous xenon lamp
anu a 7 W pulseu xenon lamp, fuinisheu with a 176-uS2-QS Bellma (Bellma
Bispania, Baicelona, Spain) flow cell with an innei volume of 18 µL, was
useu to monitoi fluoiescence uetection. A uilson (villieis-le-Bel, Fiance)
Ninipuls S low-piessuie peiistaltic pump anu 0mnifit (Cambiiuge, 0K)
Teflon tubing of u.S mm i.u. weie also useu foi constiucting the post-
column ueiivatisation manifolu. Chiomatogiaphic sepaiation was
peifoimeu using an 0nyx monolithic C18 column (Phenomenex, Toiiance,
0SA), 1uu mm x 4.6 mm i.u., poie sizes: mesopoies (1S nm), maciopoies (2
µm).

1.2. Reagents

All chemicals useu weie of analytical ieagent giaue. Stock solutions
(Suuu mg¡mL) of the flavonoius queicetin (Q0E) (Aluiich), kaempfeiol
(KAE) (Sigma), naiingin (NAR) (Fluka), naiingenin (NAN) (Aluiich) anu
hespeiiuin (BES) (Fluka) weie piepaieu in absolute ethanol. Inteimeuiate
solutions of 1uu mg¡mL weie also piepaieu by uiluting the stock solutions
in absolute ethanol. These solutions weie stoieu at 4 ºC in the uaik anu
weie stable foi at least two weeks. Woiking stanuaiu solutions weie
piepaieu fiom the inteimeuiate solutions by theii uilution in uistilleu
watei.

The mobile phase foi the chiomatogiaphic sepaiation was
constituteu by a teinaiy mixtuie of u.1S N acetic aciu, pB 4.u (solvent A),
acetonitiile (ACN) (solvent B), anu methanol (solvent C), which weie mixeu
opeiating in giauient moue uuiing the chiomatogiaphic sepaiation. All
solvents weie of analytical oi BPLC giaue (PanieacQuimica, S.A., Baicelona,
Spain). The ueiivatization ieagent solution containeu teibium(III) (S.u
mN), fiom teibium(III) nitiate pentahyuiate (Aluiich), aluminium (III)
(1S.u mN), fiom aluminium (III) nitiate (Neick) anu u.1 mN souium
Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

119
uouecylsulfate (SBS) (Neick) uissolveu in u.1S N BAc¡NaAc buffei of pB =
4.u. Ethyleneuiaminetetiaacetic aciu (EBTA) (Fluka), n-tiioctylphosphine
oxiue (T0P0) (Sigma), cetyltiimethyl ammonium biomiue (CTAB) (Sigma)
anu Tiiton X-1uu (Fluka) weie also useu.

1.3. Manifold and Procedure

Figure 2 shows the integiateu LC
sepaiation¡ueiivatization¡uetection appioach. Stanuaius oi uiluteu
samples (2uu µL), containing a mixtuie of the analytes at concentiations
within theii coiiesponuing uynamic ianges, weie injecteu in tiiplicate into
the column. The mobile phase was pumpeu at a flow-iate of u.7 mL¡min
anu the system opeiateu in an optimizeu giauient moue in oiuei to obtain
the auequate sepaiation of the analytes.
Aftei the chiomatogiaphic sepaiation, the elution solution was
meigeu with the ueiivatizing solution, which was pumpeu at a flow-iate of
u.S mL¡min. The mixtuie passeu thiough the ieaction coil, in which the
ueiivatizing ieactions took place, anu the luminiscence intensity of each
analyte was monitoieu at i
ex
S6u anu i
em
S4S nm foi 2S min.
Chiomatogiams weie taken using the oiiginal softwaie of the luminescence
spectiometei anu the iaw uata of luminescence intensity anu time weie
expoiteu anu tieateu using auequate softwaie packages foi the estimation
of the main chiomatogiaphic paiameteis.


1.4. Analysis of orange juice samples

Commeicial oi natuial oiange juice samples weie uiluteu, about
Suu-times, with uistilleu watei to match the lineai ianges of calibiation foi
each analyte anu weie uiiectly injecteu onto the chiomatogiaphic system
following the pioceuuie above inuicateu. Each ueteimination was the mean
of thiee measuiements.






Capítulo 2

120














Figure 2. Integiateu sepaiation-ueiivatization anu uetection appioach. A, B anu C
uenote 1S.u mN acetic aciu, acetonitiile anu methanol solutions,
iespectively; SB, solvent ueliveiy system; BPP, Bigh-piessuie
quateinaiy giauient pump; BPIv, Bigh Piessuie injection valve; C
18
NC,
C
18
monolithic column; BR, ueiivatizing ieagent solution; LPP, low-
piessuie pump; L, mixing ieactoi; FLB, fluoiescence uetectoi; PC,
peisonal computei; w, waste.


2. Results and discussion

2.1. Study of the luminescence reaction

Flavonoius have stiong cooiuination ability towaiu a wiue iange of
cations such as iion(II) |16j, chiomium(III) |17j, aluminium(III) |14, 18-
21j, anu lanthaniue |1S, 22, 2Sj ions, which is asciibeu to the piesence of
multiple hyuioxyl gioups anu the caibonyl gioup on the heteiocyclic iing
of theii chemical stiuctuie. Expeiimental stuuies ieveal that the piefeiieu
complexation sites foi the flavonoius involve the hyuioxyl gioup on caibon
S oi S anu the aujacent 4-caibonyl gioup |18-21j. Aluminium(III) anu
lanthaniue ions such as teibium(III) foim fluoiescent chelates with these
compounus, which have uiffeient fluoiescent featuies. While aluminium
chelates show the wiue emission banu coiiesponuing to moleculai
fluoiescence, teibium chelates show the typical naiiow emission banus of
Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

121
sensitizeu luminescence, which involves an intiamoleculai eneigy tiansfei
piocess fiom the liganu to the teibium(III) ion anu the emission of the
lanthaniue ion |1Sj.

Seveial assays weie caiiieu out to stuuy the fluoiescent behavioi of
the analytes using both teibium(III) anu aluminium(III) ions, in oiuei to
obtain a potential inciease on the luminescence signals. Figure 3 shows the
excitation anu emission spectia obtaineu foi queicetin in the piesence of
SBS as sub-micellai meuium. The excitation spectia (Figure 3.A) show a
banu with maximum excitation wavelength at S6u nm, which coiiesponus
to the teibium quelate, anu anothei moie intense banu at 4Su nm,
coiiesponuing to the aluminium chelate. Bowevei, in the piesence of both
teibium(III) anu aluminium(III), the intensity at S6u nm incieases, wheieas
that at 4Su nm uecieases. The emission spectia obtaineu by exciting at S6u
nm (Figure 3.B) show the naiiow emission banus at 49u, S4S, S8S anu 62u
nm, coiiesponuing to the teibium sensitizeu luminescence. As can be seen,
the intensity of these banus incieases when both teibium(III) anu
aluminium(III) aie togethei, obtaining the highest signal at S4S nm.
Bowevei, using 4Su nm as the excitation wavelength, the emission spectia
(Figure 3.C) show a wiue banu coiiesponuing to the emission of the
aluminium(III) chelate, which uecieases in the piesence of teibium(III). A
potential explanation of the inciease on the intensity of the teibium(III)
emission in the piesence of aluminium(III) coulu be asciibeu to the co-
luminescence effect, in which the aluminium chelate contiibutes to
impiove the teibium(III) luminescence |1Sj.

The special luminescent behavioi of the analytes in the piesence of
both aluminium(III) anu teibium(III) ions has been exploiteu to uevelop a
new post-column ueiivatization system, using an auequate mixtuie of these
ions in a sub-micellai meuium integiateu by SBS, monitoiing the
luminescence intensity of each chiomatogiaphic banu at i
ex
S6u anu i
em

S4S nm.






Capítulo 2

122































Figure 3. Excitation anu emission spectia of 1uu ng¡mL queicetin uissolveu in a
mixtuie containing the initial mobile phase composition anu u.1 N
SBS. (A) excitation (B) emission spectia of the following solutions: (1)
blank solution, (2) S mN teibium (III), (S) 1S mNaluminium (III) anu
(4) a mixtuie containing 1S mN of aluminium anu S mN of teibium
solutions (foi uetail see the text).

300 350 400 450 500
0
500
1000
2000
3000
4000
3
4
1
Excitationwavelenght, nm ( λ
em
= 545 nm)
2
(A)

300 350 400 450 500
0
500
1000
2000
3000
4000
3
4
1
2

400 500 550 600 650
0
500
1000
2000
3000
4000
4
3
1
Emissionwavelenght, nm ( λ
2
(C)



400 500 550 600 650
0
500
1000
2000
3000
4000
4
3
1

Emissionwavelenght, nm (
ex
= 450 nm)
2
(C)





0
500
1000
2000
3000
4000
4
3
1
λ
ex
= 360 nm)
2
(B)
400 450 500 550 600 650


0
500
1000
2000
3000
4000
4
3
1
Emissionwavelenght, nm (
(B)
400 450 500 550 600 650
L
u
m
i
n
e
s
c
e
n
c
e

I
n
t
e
n
s
i
t
y
,

a
.
u
.

Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

123
2.2. Optimization of Variables

The vaiiables affecting the methou weie optimizeu using the
univaiiate methouology. Table 1 shows the iange stuuieu foi each vaiiable
anu the values chosen, which weie those yieluing maximum luminescence
signals with minimum stanuaiu ueviations.

Table 1. 0ptimization of vaiiables

Variables

Variables
Range
studied
Optimum
value
Instrumental
variables

Excitation wavelength,
nm
Emission wavelength, nm
Ex/em slits
PMT gain, V

200 – 600
400 – 600
2.5 – 15
400 - 800
360
545
10/10
500
Chromatographic
variables
HPP flow-rate, mL/min
Injection volume, µL
Elution mode

Temperature, ºC

[HAc], M
pH
0.5 – 3.0
20 – 500
Gradient

15 – 35

0.05 – 0.25
2.5 – 6.0
0.7
200
-

25º C

0.15
4.0
Derivatizing
variables
LPP flow-rate, mL/min
L
1
reactor length, cm

[Al(III)], mM
[Tb(III)], mM
[HAc], M
pH
[SDS], mM

0.2 – 1.5
50 – 300

0.2 -20.0
0.1 – 10.0
0.05 – 0.25
1.5 – 6.0
0.01 – 0.5

0.5
100

15.0
5.0
0.15
4.0
0.1






Capítulo 2

124
2.2.1. Chromatographic variables

Chiomatogiaphic vaiiables weie optimizeu in oiuei to obtain
acceptable iesolution of the analytes in the lowest sepaiation time. The
composition anu influence of the mobile phase was stuuieu using teinaiy
mixtuies integiate by u.1S N acetic aciu, pB 4.u (A), ACN (B) anu methanol
(C). In all cases, the methanol amount iemaineu constant, in a peicentage of
2u %. Binaiy mixtuies of acetic aciu (u.1S N, pB 4.u) anu ACN weie
assayeu, using uiffeient giauient anu isociatic piofiles, at the optimum
flow-iate of u.7 mL¡min. A lineai giauient between 1u to 2u % of ACN in 12
min pioviues the sepaiation of the flavanone-0-glycosiues naiingin anu
hespeiiuin. The seconu pait is a giauient zone in which the ACN peicentage
is iapiuly ieuuceu to u %, followeu of an inciease to 4u %, which allows the
elution of queicetin befoie 17 min. Finally, an isociatic segment is useu to
sepaiate naiingenin anu kaempfeiol.

The influence of the chiomatogiaphic flow-iate was stuuieu in the
iange u.S-S mL¡min with acceptable sepaiation in all instances. It was
necessaiy to make compatible the flow iate with the mobile-phase
composition, in oiuei to obtain a goou peak iesolution, anu the sepaiation
piocess with the post-column ueiivatization system. A flow-iate of u.7
mL¡min was acceptable as it geneiates piessuies lowei than the maximum
piessuie toleiateu in the luminescence uetectoi.

2.2.2. Post-column derivatization variables

The stuuy of the flow-iate of the ueiivatizing ieagent solution
showeu that u.S mL¡min pioviueu an auequate flow-iate to the optimum
ueiivatizing ieactions when u,7 mL¡min was useu foi the chiomatogiaphic
sepaiation. The length of the mixtuie ieactoi L was 1uu cm, which was
enough foi the uevelopment of these ieactions, uue to the ielatively fast
iate foimation of the aluminum(III) anu teibium(III) chelates.

The luminescence of lanthaniue chelates can be quencheu by the
vibiational eneigy of the hyuioxyl gioups of watei molecules |1Sj, which is
ieally notable when ieveise-phase LC is useu uue to the ielative high
content of watei in the mobile phase. It has been pieviously uesciibeu that
an auequate luminescent signal can be obtaineu in the piesence of acetate
Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

125
ions |24, 2Sj, which can be asciibeu to theii capability to uisplace watei
fiom the fiist cooiuination spheie of teibium(III). A souium acetate¡acetic
aciu buffei solution was chosen to keep the auequate pB in the final
solution anu ieach the highest luminescence signal by the positive effect of
acetate ions. The best acetic aciu concentiation was u.1S N, being the pB of
the ueiivatization solution aujusteu to 4.u.

Figure 4 uepicts the effect of thiee chemical vaiiables on the
luminescence of the flavanoius chelates. The influence of teibium(III)
concentiation was evaluateu in the iange u.1 to 1u.u mN (Figure 4.A),
choosing a concentiation of S.u mN as optimum. Accoiuing to the stuuy of
the influence of aluminium(III) concentiation (Figure 4.B), a value of 1S.u
mN was chosen. The potential positive effect of a suifactant such as Tiiton
X-1uu, CTAB anu SBS was also stuuieu. The luminescence of the flavanoius
chelates was not mouifieu by the piesence of the two fiists, but it incieaseu
in the piesence of SBS, as Figure 4.C shows. A u.1 mN SBS concentiation
was selecteu. In oiuei to impiove the luminescence signal,
tiioctylphosphine oxiue anu EBTA, as effective luminescence syneigetic
agents, weie testeu, but not changes in the luminescence signal weie
obtaineu.

2.3. Analytical features

Calibiation giaphs weie iun unuei the optimal expeiimental
conuition uepicteu in Table 1. Seven stanuaiu solutions of mixtuies of the
five analytes, coveiing the concentiation iange between 1 anu Suuu ng¡mL,
weie assayeu in tiiplicate. Table 2 summaiizes the figuies of meiits of the
methou, incluuing aveiage ietention times, calibiation equations,
iegiession coefficients, iesiuual stanuaiu ueviations, lineai ianges,
uetection limits (L0Bs) anu piecision, expiesseu as ielative stanuaiu
ueviation (RSB %) values. The L0Bs weie calculateu accoiuing I0PAC
iecommenuations |26j. As can be seen, the methou was enough sensitive
foi the ueteimination of the analytes at the ng¡mL level, giving wiue
uynamic ianges, between 2.1 anu 2uuu ng¡mL, uepenuing on the analyte
selecteu. The iegiession values (i
2
≥ u.99S6, n = 7) anu iesiuual stanuaiu
ueviation (σ
y¡x
≤ u.9S) coefficients weie acceptable in all instances.


Capítulo 2

126

































Figure 4.Influence of Tb(III) (A), Al(III) (B) anu SBS (C) concentiations on the
luminescence of naiingin (1), hespeiiuin (2), queicetin (S), naiingenin
(4) anu kaempfeiol (S). |Analytej = 1uu ng¡ml. |Al(III)j = 2 mN (A) anu
1S mN (C). |Tb(III)j = S mN (B anu C). |SBSj = u.uS mN (A anu B)

Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

127
The piecision, expiesseu as peicentage of the ielative stanuaiu
ueviation was establisheu in two chaiacteiistic zones coiiesponuing to the
minimal (centioiu point) anu maximal (neaily of the aveiage of the L0Qs)
eiiois of the uynamic calibiation giaphs. The RSB % values, estimateu foi n
= 11 solutions containing Suu anu 1u ng¡mL of each analyte, iespectively,
injecteu in tiiplicate, iangeu between 1.S anu 4.7 %.

2.4. Application of the method

The pioposeu methou was applieu to the analysis of oiange juice
samples fiom natuial anu commeicial oiigin. The samples only iequiieu
the auequate uilution, about Suu-times, with uistilleu watei to match the
lineai iange of the calibiation giaph foi each analyte. Figure 5 shows the
chiomatogiams obtaineu foi a stanuaiu solution containing 1uu ng¡mL of
each analyte (Fig. 5.A), anu foi natuial (Fig. 5.B) anu commeicial oiange
juice samples (Fig. 5.C). The methou was testeu in two ways: a) by
ueteimining the concentiation of the five analytes (naiingin, hespeiiuin,
queicetin, naiingenin anu kaempfeiol) in the samples, anu b) by stuuying
the iecoveiy aftei auuition of two stanuaiu aliquots of 1u.u anu Suu.u
ng¡mL of each analyte. The samples weie analyzeu following the sampling
pioceuuie uesciibeu in the expeiimental section. The iesults achieveu aie
piesenteu in Table 3, in which can be seen that all the analytes weie founu
in the analyzeu samples, at concentiation levels similai to those uesciibeu
elsewheie |Sj, except kaempfeiol, which was not quantifieu in samples 1
anu S. The iecoveiies iangeu between 86.9 anu 1u8.2 % in all instances,
which was inuicative of the low matiix inteifeience.












T
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y
.

Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

129
































Figure 5. Chiomatogiams achieveu using the pioposeu appioach. (A) An aqueous
stanuaiu solution containing 1uu ng¡mL of each analyte, (B) natuial
anu commeicial (C-B) juice samples.Analytes: (1) naiingin, (2)
hespeiiuin, (S) queicetin, (4) naiingenin anu (S) kaempfeiol.
Capitulo 2
130
3. Conclusions

This aiticle shows the usefulness of a new post-column
ueiivatization ieaction foi the LC sepaiation anu luminescent uetection of
flavonoius in oiange juices. It is the fiist time that the simultaneous
foimation of analyte chelates with two metal ions to obtain sensitizeu
luminescence is uesciibeu. 0nlike othei LC methous foi the analysis of
similai samples, which involve a pievious sample tieatment |6-1uj, the
pioposeu methou allows the uiiect injection of the uiluteu samples. Also,
the time iequiieu foi the chiomatogiaphic sepaiation is shoitei than that
iequiieu foi the sepaiation of similai mixtuies of these compounus |7-9j.
Finally, iegaiuing L0B values, the luminescent uetection impiove the
values pieviously uesciibeu using photometiic, coulometiic oi NS
uetection |8, 1uj.


Acknowledgements

Authois giatefully acknowleuge financial suppoit fiom the Spanish
NCyT (Ninisteiio ue Ciencia y Tecnologia) (uiant No. CTQ2uu9-
u8621¡BQ0) anu the }unta ue Anualucia (uiant No. P06-FQN-u1SS6).

















Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

131
References

|1j Chu, Y.H.; Chang, C.L.; Hsu, H.F.; J. Sci. Food Agricul. 2000,
80, 561-566.
|2j Scalbeit, A.; Nanach, C.; Noianu, C.; Rémésy, C.; }iménez, L.; Ciit.
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|Sj Liu, E.B.; Qi, L.W.; Cao, }.; Li, P.; Li, C.Y.; Peng, Y.B.; Nolecules 2uu8,
1S, 2S21-2S44.
|4j Aheine, S.A.; 0'Biien, N.N.; Nutiition 2uu2, 18, 7S-81.
|Sj uattuso, u.; Baiieca, B.; uaigiulli, C.; Leuzzi, 0.; Caiisti, C.; Nolecules
2uu7, 12, 1641-167S.
|6j }ustesen, 0.; Knuthsen, P.; Leth. T.; }. Chiomatogi. A, 1998, 799 1u1-
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|7j Nouly, P.; uayuou, E.N.; Auffiay, A.; }. Chiomatogi. A, 1998, 8uu,
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|8j Belajová, E.; Suhaj, N.; Foou Chem. 2uu4, 86 SS9-S4S.
|9j Abau-uaicia, B.; Beiiueta, L.A.; Lopez-Náiquez, B.N.; Ciespo-Feiiei,
I.; uallo, B.; vicente, F.; }. Chiomatogi. A, 2uu7, 11S4 87-96.
|1uj Caieii, N.; Elviii, L.; Nangia, A.; Nusci, N.; }. Chiomatogi. A, 2uuu,
881, 449-46u.
|11j Bugo, P.; Lo-Piesti, N.; 0hman, N.; Fazzio, A.; Bugo, u.; Nonuello, L.;
}. Sep. Sci. 2uuS, 28, 1149-11S6.
|12j Füzfai, Z.; Nolnái-Peil, I.; }. Chiomatogi. A, 2uu7, 1149, 88-1u1.
|1Sj Russo, P.; Anuieu-Navaiio, A.; Aguilai Caballos, N.P.; Feinánuez-
Romeio, }.N.; uomez-Bens, A.; }. Agiic. FoouChem. 2uu8, S6, 18S8-
186S.
|14j Bollman, P.C.B.; van Tiijp, }.N.P.; Buysman, N.N.C.; Anal. Chem.
1996, 68, SS11-SS1S.
|1Sj uomez-Bens, A.; Aguilai-Caballos, N.P.; Tienus Anal. Chem. 2uu2,
21, 1S1-141.
|16j Leopoluini, N.; Russo, N.; Chiouo, S.; Toscano, N.; }. Agiic. Foou
Chem. 2uu6, S4, 6S4S-6SS1.
|17j Chen, W.; Sun, S.; Cao, W.; Liang, Y.; Song, }.; }. Nol. Stiuct. 2uu9, 918,
194-197.
|18j Bouuet, A.C.; Coinaiu, }.P.; Neilin, }.C.; Spectiochim. Acta A 2uuu, S6,
829-8S9
|19j Castio, u.T.; Blanco, S.E.; Spectiochim. Acta A 2uu4, 6u, 22SS-2241.
|2uj Beng, B.; van Beikel, u.}.; }. Nass Spectiom. 1998, SS, 1u8u-1u87.
Capitulo 2
132
|21j Smith, u.}.; Thomsen, S.}.; Naikham, K.R.; Anuaiy, C.; Caiuon, B.; }.
Photochem. Photobiol. 2uuu, 1S6, 87-91.
|22j Zhou, }.; uong, u.Q.; Zhang, Y.N.; Qu, }.Q.; Wang, L.F.; Xu, }.X.; Anal.
Chim. Acta. 1999, S81 17-22.
|2Sj Shaghaghi, N.; Nanzooii, }.L.; }ouyban, A.; Fouu Chem. 2uu8, 1u8,
69S-7u1.
|24j Rouiiguez-Biaz, R.C.; Feinánuez-Romeio, }.N.; Aguilai-Caballos,
N.P.; uomez-Bens, A.; Anal. Chim. Acta. 2uu6, S78, 22u-226.
|2Sj Rouiiguez-Biaz, R.C.; Feinánuez-Romeio, }.N.; Aguilai-Caballos,
N.P., uomez-Bens, A.; }. Agiic. Foou Chem. 2uu6, S4, 967u-9676.
|26j Long, u.L.; Winefoiunei, }.B.; Anal. Chem. 198S, SS, 712-717.

Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

133

Chiomatogiaphia 72 (11-12), pp. 111S-112u


Long-Wavelength Fluorescence detection of
Flavonoids in Orange Juices using Liquid
Chromatography


A. Andreu-Navarro, J.M. Fernández-Romero, A. Gómez-Hens
Bepaitment of Analytical Chemistiy, Faculty of Sciences, 0niveisity of
Coiuoba, "Naiie Cuiie Annex" Builuing. Campus of Rabanales, E-14u71
Coiuoba, Spain


A new post-column liquiu chiomatogiaphic ieaction system foi the
ueteimination of flavonoius in oiange juices is baseu on the use of the long
wavelength fluoiophoi ciesyl violet anu ceiium(Iv) in a cetyl
tiimethylammonium biomiue micellai meuium. Two flavone aglycones
(queicetin anu kaempfeiol), a flavonone aglycone (naiingenin), one
flavone-0-glycosiue (iutin) anu two flavanone-0-glycosiues (hespeiiuin
anu naiingin) have been useu as analyte mouels. The ieaction piocess
involves the inteiaction between the analyte, ceiium(Iv) anu ciesyl violet
giving iise to a ueciease in the fluoiescence, measuieu at i
ex
S8S, i
em
62S
nm, which is piopoitional to the analyte concentiation. Bynamic ianges of
the calibiation giaphs anu uetection limits, obtaineu with stanuaiu
solutions of the analytes aie (ng mL
-1
): queicetin (12.2 - 4uuu, S.7),
kaempfeiol (S.S - 1uuu, 1.u), naiingenin (6.7 - 1uuu, 2.u), iutin (S.u - 8uu,
1.S), hespeiiuin (1u.1 - 1uuu, S.u), anu naiingin (17.8 - 8uu, 1.8). The
ueteimination coefficients weie highei than u.99S in all instances. The
piecision of the methou, expiesseu as RSB%, was establisheu at two
concentiation levels, with values ianging between 2.8 anu 6.2 %. The
piactical usefulness of the uevelopeu methou is uemonstiateu by the
Capítulo 2
134
analysis of natuial anu commeicial oiange juices, which weie filteieu,
uiluteu anu uiiectly injecteu into the chiomatogiaphic system, with
appaient iecoveiies between 86.9 anu 1u7.u%.






































Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

135
Introduction

The use of long-wavelength fluoiophoies (LWF) as analytical
ieagents is an attiactive option to impiove the selectivity of fluoiimetiic
analysis anu avoiu oi minimize the sample tieatment step of the analytical
piocess. These uyes allow measuiements aie obtaineu in a iegion of the
electiomagnetic spectium (> 6uu nm) in which the potential absoiption oi
emission associateu to sample matiix is minimizeu. Also, Raman
inteifeience is gieatly uiminisheu anu the piobability of non-iauiative
quenching piocesses is uecieaseu uue to the usually shoit fluoiescence
lifetime of these fluoiophoies. LWFs have been uesciibeu as enzymatic
substiates, immunoassay labels, sensing systems anu ueiivatizing ieagents
in liquiu chiomatogiaphy (LC) anu capillaiy electiophoiesis (CE) |1j.
Although the lack of sufficient ieactive gioups foi taigeting of analytes is a
limitation of these fluoiophoies, theii analytical usefulness has been
expanueu using electiostatic anu ieuox inteiactions |2-6j.

This aiticle uesciibes foi the fiist time the use of the oxazine LWF
ciesyl violet (Cv) in a post-column LC ieaction system foi the inuiiect
ueteimination of six iepiesentative flavonoius in oiange juices, involving
the use of ceiium(Iv) anu a cetyl tiimethylammonium biomiue (CTAB)
micellai meuium anu fluoiescence uetection.

Flavonoius aie a laige gioup of natuial polyphenols founu in fiuits
anu vegetables that have been wiuely stuuieu by LC with photometiic oi
mass spectiometiic uetection |7j. Some of these methous have been
applieu to the analysis of oiange juice samples, iequiiing most of them a
tieatment step, which involves extiaction, hyuiolysis, centiifugation
anu¡oi filtiation |8-1Sj. A iecent uiiect LC methou uesciibeu foi this
puipose is baseu on the use of post-column luminescence ueiivatization
ieactions of the analytes with aluminium(III) anu teibium(III) |14j.
Although the uetection limits obtaineu aie similai to the values ieacheu in
this manusciipt, the time iequiieu foi the mixtuie iesolution is highei.
Also, a uiiect LC methou with photometiic uetection has been pioposeu foi
the ueteimination of a mixtuie of flavonoius, similai to that pioposeu heie
(figure 1), in oiange extiacts |1Sj but the sepaiation iequiieu SS min.


Capítulo 2
136
A) B)







A) R
1
R
2
R
S
R
4

Naiingin (Flavanone 0-glycosiue) B ulycosiue B 0B
Bespeiiuin (Flavanone 0-glycosiue) B ulycosiue 0B 0CB
S

Naiingenin Flavanone aglycone) B B B 0B
B)
Rutin (Flavone 0-glycosiue) ulycosiue 0B 0B 0B
Queicetin (Flavone 0-aglycone) 0B B 0B 0B
Kaemfpheiol (Flavone aglycone) 0B B B 0B

Figure 1. Chemical stiuctuies anu nomenclatuie of the flavonoius selecteu in this
ieseaich.



1. Experimental

1.1. Nateiials

All chemicals useu weie of analytical ieagent giaue. Stock solutions
(Suuu mg L
-1
) of the analytes weie piepaieu as follows: queicetin (Q0E)
(Aluiich), iutin (R0T) (Sigma), kaempfeiol (KAE) (Sigma), naiingin (NAR)
(Fluka), naiingenin (NAN) (Aluiich) anu hespeiiuin (BES) (Fluka) weie
uissolveu in absolute ethanol. Inteimeuiate solutions of 1uu mg L
-1
weie
also piepaieu by uiluting the stock solutions in absolute ethanol oi watei,
accoiuing to the analyte solubility. Stock anu inteimeuiate solutions weie
stoieu at 4 ºC in the uaik anu weie stable foi at least two weeks. Woiking
stanuaiu solutions weie piepaieu fiom the inteimeuiate solutions by theii
uilution in uistilleu watei.




O
O
R
2
O
O H
R
1
R
3
R
4
O
O
R
2
O
O H
R
1
R
3
R
4
O
O
R
2
O
O H
R
1
R
3
R
4
O
O
R
2
O
O H
R
1
R
3
R
4
O
O
R
2
O
O H
R
1
R
3
R
4
Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

137
1.2. Equipment

HPLC. An Agilent 12uu Seiies Liquiu Chiomatogiaphy System
composeu by a quateinaiy pump, a uegassei unit, a vial autosamplei, anu a
theimostateu column compaitment has been useu. Chiomatogiaphic
sepaiation was peifoimeu using an 0nyx monolithic C18 column
(Phenomenex, Toiiance, 0SA), 1uu mm x 4.6 mm i.u., poie sizes:
mesopoies (1S nm), maciopoies (2 µm).

Post-column reaction system. An SLN Aminco (0ibana, IL) AB2
luminescence spectiometei pioviueu with a 1Su W continuous xenon lamp,
fuinisheu with a 176-uS2-QS Bellma (Bellma Bispania, Baicelona, Spain)
flow cell with an innei volume of 18 µL, was useu to monitoi fluoiescence
uetection. A uilson (villieis-le-Bel, Fiance) Ninipuls S low-piessuie
peiistaltic pump anu 0mnifit (Cambiiuge, 0K) Teflon tubing of u.S mm i.u.
weie also useu foi constiucting the post-column ieaction manifolu. The
ieaction solution was also pieviously meigeu fiom two sepaiateu solutions
containing the ieactants (L
1
= Su cm). The mixtuie passeu thiough the
ieaction coil (L
2
= 1uu cm), in which the ieaction piocess took place, anu
the fluoiescence intensity was monitoieu using i
ex
S8S, i
em
62S nm.

1.S. Nethou

Figure 2 shows the integiateu LC sepaiation¡ieaction¡uetection
appioach. Stanuaius oi uiluteu samples (2uu µL), containing a mixtuie of
the analytes at concentiations within theii coiiesponuing uynamic ianges,
weie injecteu in tiiplicate into the column. The mobile phase was pumpeu
at a flow-iate of u.7 mL min
-1
anu the system opeiateu in an optimizeu
giauient moue in oiuei to obtain the auequate sepaiation of the analytes.
The mobile phase was constituteu by a teinaiy mixtuie of u.1S mol L
-1
acetic buffei, pB 4.u (solvent A), acetonitiile (solvent B), anu methanol
(solvent C), which weie mixeu opeiating in giauient moue uuiing the
chiomatogiaphic sepaiation (Table 1). All solvents weie of analytical oi
BPLC giaue (Panieac Quimica, S.A., Baicelona, Spain). The ieaction solution
was meigeu in a continuous system by mixing two solutions: solution 1,
containing ciesyl violet (Cv) (8.S µmol L
-1
) (Sigma) anu cetyltiimethyl
ammonium biomiue (CTAB) (1 mmol L
-1
) (Sigma) uissolveu in watei, anu
Capítulo 2
138
solution 2, containing ceiium(Iv) sulfate (u.1 mmol L
-1
) (Neick) uissolveu
in 1S mmol L
-1
sulfuiic aciu.




















Figure 2. Integiateu sepaiation-ueiivatization anu uetection appioach. A, B anu C
uenote u.1S mol L
-1
acetic buffei, acetonitiile anu methanol solutions,
iespectively; SB, solvent ueliveiy system; BPP, Bigh-piessuie quateinaiy
giauient pump; BPIv, Bigh piessuie injection valve; C
18
NC, C
18

monolithic column; B1, Cv in CTAB micellai solution; B2, ceiium(Iv) in
sulfuiic aciu solution; LPP, low-piessuie pump; L
1
anu L
2
, mixing
ieactois; FLB, fluoiescence uetectoi; PC, peisonal computei, w, waste
solution.


Table 1. Elution giauient conuitions

Time (min)
% Acetic buffer
(0.15 mol L
-1
, pH 4.0)
% Acetonitrile % Methanol
u 7S S 2u
S 7u 1u 2u
12 Su Su 2u
1S Su Su 2u

FIGURE 2
LPP
D1
w
2
w
1
C
18
MC
A C
HPP
SD
HPIV
L2
PC
FLD
B
L1
D2
Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

139
Aftei the chiomatogiaphic sepaiation, the elution solution was
meigeu with the ieaction solution, which was pumpeu at a flow-iate of u.9
mL min
-1
. Chiomatogiams weie taken using the oiiginal softwaie of the
spectiofluoiimetei anu the iaw uata of fluoiescence intensity anu time
weie expoiteu anu tieateu using suitable softwaie packages foi the
estimation of the main chiomatogiaphic paiameteis.

1.4. Analysis of oiange juice samples

Commeicial anu natuial oiange juice samples weie filteieu anu
uiluteu Suu-times with uistilleu watei, to match the lineai iange of the
calibiation giaph foi each analyte anu weie uiiectly injecteu onto the
chiomatogiaphic system following the pioceuuie above inuicateu. Each
ueteimination was the mean of thiee measuiements.


2. Results and discussion

2.1. Stuuy of the fluoiescent system

Figure 3 shows the emission spectia (λ
ex
S8S nm) of Cv alone anu
in the piesence of the uiffeient components of the system, using queicetin
as a flavonoiu mouel. The spectia weie obtaineu in a solution containing
the initial mobile phase composition (7S% acetic buffei, S% acetonitiile
anu 2u% methanol) useu foi the chiomatogiaphic sepaiation. As can be
seen, the fluoiescence of Cv (cuive 1) shows a slight inciease in the
piesence of CTAB micelles (cuive 2), in a similai way to the behavioui
pieviously uesciibeu foi its absoibance spectium |16j. The oxiuation of Cv
by ceiium(Iv), which is maue eviuent by the uiastic fluoiescence
quenching (cuive S), is piactically hinueieu in the piesence of CTAB (cuive
4) because the uye is piotecteu by the micelles anu only a paitial ueciease
of the fluoiescence occuis. Bowevei, the piesence of queicetin causes a
quenching in the Cv fluoiescence (cuives S anu 6), which is piopoitional to
the flavonoiu concentiation.




Capítulo 2
140
















Figure 3. Emission spectia (λ
ex
S8S nm) of (1) Cv alone anu (2) in the piesence of
CTAB, (S) ceiium(Iv), (4) CTAB + ceiium(Iv), (S) CTAB + ceiium(Iv) +
queicetin (2u ng mL
-1
), (6) anu CTAB + ceiium(Iv) + queicetin (1uu ng
mL
-1
). All spectia weie obtaineu in the piesence of a solution containing
7S% acetic buffei (u.1S mol L
-1
), S% acetonitiile anu 2u% methanol,
which coiiesponus to the initial mobile phase composition. |Cvj = 8.S
µmol L
-1
, |CTABj = 1.u mmol L
-1
, ceiium(Iv) = u.1 mmol L
-1
.


This fluoiescence quenching is piobably the iesult of a combination
of ieuox, aciu-base anu hyuiophobic piocesses. It has been uesciibeu that
queicetin has a stiong inteiaction with CTAB micelles, which mouifies theii
stiuctuie |17j. Thus, the ueciease in the fluoiescence of Cv coulu be
asciibeu to the inteiaction of the flavonoiu with the micelles, which
facilitates that the uye leaves the micelles anu is oxiuizeu by ceiium(Iv). It
is also logical to assume that the flavonoiu can be oxiuizeu by ceiium(Iv)
befoie its inteiaction with CTAB micelles. In fact, it has been uesciibeu that
CTAB micelle facilitates the oxiuation of queicetin |17j. 0n the othei hanu,
although the uissociation constants of queicetin aie ielatively high, anu the
appaient pB of the meuium is about S.8, the CTAB micelles can pioviue a
ielative alkaline micioenviionment |18j. This meuium can piomote the
foimation of queicetin anions, giving iise to an auuitional electiostatic
attiaction with positively chaigeu CTAB micelle, besiues the hyuiophobic
inteiaction.
600 620 640 660 680 700
0
400
800
1200
1600
2000
F
l
u
o
r
e
s
c
e
n
c
e

i
n
t
e
n
s
i
t
y

Emission wavelength (nm) (λ
ex
= 585 nm)
(3)
(1)
(2)
(4)
(5)
(6)
Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

141
2.2. Nethou Peifoimance

The hyuiouynamic anu chemical vaiiables affecting the system
weie optimizeu using the univaiiate methou. Table 2 shows the iange
stuuieu foi each vaiiable anu the values chosen, which weie those yieluing
the best uiffeiences between the blank solution anu the solution containing
the analytes, anu with minimum stanuaiu ueviation.


Table 2. 0ptimization of vaiiables

Type of Variables

Variables
Range
studied
Optimu
m value
Instiumental
Excitation wavelength, nm
Emission wavelength, nm
Ex¡em slits
PNT gain, v
Suu - 62u
61u - 8uu
2.S - 1S
4uu - 8uu
S8S
62S
8¡8
6uu
Chiomatogiaphic
BPP flow-iate, mL min
-1
Injection volume, µL
Elution moue

Tempeiatuie, ºC
|BAcj mol L
-1

pB
u.S - S.u
2u - Suu
uiauient

2S.u - 4S.u
u.uS - u.2S
2.S - 6.u
u.7
2uu
-

SS.u
u,1S
4.u
Beiivatizing
LPP flow-iate, mL min
-1

L
1
ieactoi length, cm
L
2
ieactoi length, cm

|Ciesyl violetj µmol L
-1

|Ce (Iv)j mmol L
-1

|B
2
S0
4
j mmol L
-1

|CTABj mmol L
-1

u.2 - 1.S
Su - Suu
Su - Suu

1.u -2u.u
u.uS - u.2
S.u - Su.u
u.u1 - 2.u
u.9
Su
1uu

8.S
u.1u
1S.u
1.u


2.S. Chiomatogiaphic sepaiation

Chiomatogiaphic vaiiables weie optimizeu in oiuei to obtain
acceptable iesolution of the analytes in the lowest sepaiation time. The
composition anu influence of the mobile phase was stuuieu using teinaiy
mixtuies integiateu by u.1S mol L
-1
acetic buffei, pB 4.u (A), acetonitiile
(B) anu methanol (C). Biffeient giauient piofiles weie assayeu at the
optimum flow iate, finuing that the use of a constant methanol peicentage
Capítulo 2
142
(2u%) anu incieasing peicentages of acetonitiile until Su %, with the
simultaneous uecieasing of the acetic buffei, up to 12 min (Table 1), was
useful foi the iesolution of the analytes.

The influence of the chiomatogiaphic flow-iate was stuuieu in the
iange u.S - S mL min
-1
with acceptable sepaiation in all instances. Although
monolithic columns allow the use of high flow-iates, it was necessaiy to
choose a ielatively low flow-iate, u.7 mL min
-1
, to make compatible the
sepaiation piocess with the post-column ieaction system anu the piessuie
toleiateu by the fluoiescence uetectoi.

2.4. Post-column ieaction

The stuuy of the flow-iate of the ieaction ieagent solution showeu
that u.9 mL min-1 was a suitable value foi the piocess when a u.7 mL min-1
flow-iate was useu foi the chiomatogiaphic sepaiation. The length of the
mixtuie ieactoi L2 was 1uu cm, which was enough foi the uevelopment of
the uiffeient ieactions involveu in the piocess. Now that a sensitive
iesponse has been uemonstiateu, futuie optimization of the uesign anu
connections in the post-column ieactoi may be possible to ieuuce the peak
wiuths.

Figure 4 shows the influence of Cv, ceiium(Iv) anu CTAB
concentiation on the fluoiescence of the system in the piesence of iutin
(cuive 1), naiingin (cuive 2) anu queicetin (cuive S). The values of the y-
axis coiiesponu to the net aiea of the negative peaks achieveu in each
chiomatogiam. The effect of the Cv concentiation (Figure 4a), testeu in the
iange of 1.u - 2u µmol L-1, anu in the piesence of ceiium(Iv) u.uS mmol L-
1 anu CTAB 1.u mmol L-1, was similai foi the thiee analytes. A
concentiation of 8.S µmol L-1 was selecteu as optimum. As it was
necessaiy to uissolve ceiium(Iv) in sulfuiic aciu to avoiu its piecipitation,
the effect of the concentiation of this aciu was evaluateu in the iange S.u -
Su mmol L-1, finuing that a 1S mmol L-1 concentiation pioviueu enough
aciu meuium to uissolve ceiium(Iv). The influence of ceiium(Iv)
concentiation was stuuieu in the iange u.uS - u.2 mmol L-1 (Figure 4b), in
the piesence of Cv 8.S µmol L-1 anu CTAB 1.u mmol L-1 . As can be seen,
the effect of ceiium(Iv) in the piesence of the glycosiue flavonoiu
ueiivatives iutin anu naiingin is slightly uiffeient to that obtaineu in the
Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

143
piesence of the aglycone ueiivative queicetin. A u.1u mmol L-1 ceiium(Iv)
concentiation was selecteu as optimum. The CTAB concentiation is a
ciitical vaiiable of the system. Figure 4c, obtaineu in the piesence of
ceiium(Iv) u.1 mmol L-1 anu Cv 8.S µmol L-1, shows that the maximum
signal in the piesence of iutin anu naiingin is obtaineu at a u.S mmol L-1
CTAB concentiation, which coiiesponus to the ciitical micellai
concentiation uesciibeu foi this suifactant |19j. In the piesence of
queicetin, the maximum signal is obtaineu at 1 mmol L-1, choosing this
concentiation as the optimum value.

Figure 5a shows the chiomatogiam obtaineu unuei the optimum
expeiimental conuitions foi a stanuaiu solution containing 1uu ng mL-1 of
each analyte. Although the sepaiation uoes not ieach the baseline in some
instances, it is enough to allow analyte ueteimination. The slight inciease of
the baseline is asciibeu to the vaiiation of the fluoiescence of Cv with the
solvent giauient. As can be seen, the mixtuie of the six flavonoius can be
iesolveu in about 1S min. Seveial phenolic anu hyuioxycinnamic acius,
such as gallic, caffeic anu coumaiic acius, weie testeu, but they weie co-
eluteu with the fiont of the solvent.

2.S. Analytical featuies

Calibiation giaphs weie iun unuei the optimal expeiimental
conuitions uepicteu in Table 2, anu the lineaiity of the methou was
establisheu using the iesiuual analysis test Eight stanuaiu solutions of
mixtuies of the six analytes, coveiing the concentiation iange between 1
anu 4uuu ng mL
-1
, weie assayeu in tiiplicate. Table 3 summaiizes the
figuies of meiits of the methou, incluuing aveiage ietention time,
calibiation equations, ueteimination coefficients, iesiuual stanuaiu
ueviations, lack-of-fit p-values, lineai ianges, uetection limits (L0Bs) anu
piecision. The ueteimination coefficient values (R2 >u.99S, N= 8) anu
iesiuual stanuaiu ueviation (oy¡x <1.1S) weie acceptable in all instances.






Capítulo 2
144



































Figure 4. Influence of Cv (a), ceiium(Iv) (b) anu CTAB (c) concentiations on the
fluoiescence system containing iutin (1), naiingin (2) anu queicetin (S).
|Analytej = 1uu ng mL
-1
. |ceiium(Iv)j = u.uS mmol L
-1
(a) anu u.1 mmol
L
-1
(c), |Cvj = 8.S µmol L
-1
(b anu c), |CTABj = 1.u mmol L
-1
(a anu b). (The
y-axis values coiiesponu to the absolute peak aieas achieveu foi the
selecteu analytes aftei the analytical signal coiiection fiom the blank).

(a)
0
100
200
300
400
500
600
[Cresyl violet] (µmol L
-1
)
P
E
A
K

A
R
E
A
S

(1)
(2)
(3)
1 4 7 10 13 16 19
(b)
0
200
400
600
800
1000
0.05 0.1 0.15 0.2
[Ce(IV)] (mmol L
-1
)
(1)
(2)
(3)
P
E
A
K


A
R
E
A
S

(c)
0
500
1000
1500
2000
2500
0,01 0.5 1 1.5 2
[CTAB] (mmol L
-1
)
P
E
A
K

A
R
E
A
S

Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

145


























Figure 5. Chiomatogiams achieveu using the pioposeu appioach. (a) Aqueous
stanuaiu solution containing 1uu ng mL
-1
of each analyte: iutin, (1)
naiingin, (2) hespeiiuin, (S) queicetin, (4) naiingenin (S) anu
kaempfeiol (6), anu (b) natuial oiange juice 2 (Navel) containing:
naiingin, (2) hespeiiuin, (S) queicetin, (4) anu naiingenin (S).











Time (min)
F
l
u
o
r
e
s
c
e
n
c
e

i
n
t
e
n
s
i
t
y

0
400
800
1200
1600
2000
0
400
800
1200
1600
2000
(1)
(3)
(b)
0
0
400
800
1200
1600
2000
0
400
800
1200
1600
2000
0
400
800
1200
1600
2000
0
400
800
1200
1600
2000
(2)
(4)
(5)
(a)
0 2 4 6 8 10 12
(3)
(2)
(4)
(5)
(6)
Capítulo 2
146
The lineaiity of the calibiation cuives was also estimateu using the
lack-of-fit test, which ievealeu a goou statistical coiielation between the
vaiiables in the selecteu concentiation inteivals, with p-values highei than
u.1 in all instances. The inteival of the calibiation giaphs iangeu between S
- 8uu ng mL
-1
foi iutin anu 12.2 - 4uuu ng mL
-1
foi queicetin. The L0Bs,
calculateu accoiuing I0PAC iecommenuations |2uj, iangeu between 1.u ng
mL
-1
foi kaempfeiol anu S.7 ng mL
-1
foi queicetin. The piecision of the
methou, expiesseu as peicentage of ielative stanuaiu ueviation (%RSB),
was stuuieu at two concentiations of each analyte, 2u anu 1uu ng mL
-1

(N=11), injecting each sample in tiiplicate. The values obtaineu iangeu
between 2.8 anu 6.2 %.


2.6. Application of the methou

The pioposeu methou was applieu to the analysis of five oiange
juice samples fiom natuial anu commeicial oiigin. The samples only
iequiieu the filtiation anu auequate uilution, Suu-times, with uistilleu
watei to match the lineai iange of the calibiation giaph of each analyte.
The methou was testeu in two ways: a) by ueteimining the concentiation of
the six analytes in the samples, anu b) by stuuying the iecoveiy aftei
auuition of two stanuaiu aliquots of 2u.u anu 1uu.u ng mL-1 of each
analyte. The samples weie analyzeu following the pioceuuie uesciibeu in
the expeiimental section (N= 6). The iesults achieveu aie piesenteu in
Table 4, in which can be seen that naiingin, hespeiiuin, queicetin anu
naiingenin weie founu in all the analyzeu samples. Rutin was founu in two
commeicial samples anu kaempfeiol only in one of them. Figure 5b shows
the chiomatogiam obtaineu foi the natuial oiange juice 2. The
concentiation levels founu aie similai to those uesciibeu elsewheie |8-12j.
The appaient iecoveiies iangeu between 86.9 anu 1u7.u %, which was
inuicative of the low matiix inteifeience.


T
a
b
l
e

3
.

F
e
a
t
u
i
e
s

o
f

t
h
e

m
e
t
h
o
u

(
1
)


S
B

u
e
n
o
t
e
s

s
t
a
n
u
a
i
u

u
e
v
i
a
t
i
o
n
,

(
2
)

y

u
e
n
o
t
e
s

p
e
a
k

a
i
e
a
,

x

u
e
n
o
t
e
s

a
n
a
l
y
t
e

c
o
n
c
e
n
t
i
a
t
i
o
n

(
n
g

m
L
-
1
)
,

(
S
)

i
e
s
i
u
u
a
l

s
t
a
n
u
a
i
u

u
e
v
i
a
t
i
o
n

a
n
u

p
-
v
a
l
u
e
s

a
c
h
i
e
v
e
u

u
s
i
n
g

t
h
e

l
a
c
k
-
o
f
-
f
i
t

t
e
s
t

a
n
u

(
4
)

i
e
l
a
t
i
v
e

s
t
a
n
u
a
i
u

u
e
v
i
a
t
i
o
n

(
N
=
1
1
)

v
a
l
u
e
s

a
t

2
u
.
u

a
n
u

1
u
u
.
u

n
g

m
L
-
1

o
f

e
a
c
h

a
n
a
l
y
t
e
.
A
n
a
l
y
t
e

R
e
t
e
n
t
i
o
n

t
i
m
e

±

S
D

(
m
i
n
)
(
1
)

E
q
u
a
t
i
o
n

(
2
)

R
2

y
/
x


(
p
-
v
a
l
u
e
)

(
3
)

L
i
n
e
a
r

r
a
n
g
e

(
n
g


m
L
-
1
)

D
e
t
e
c
t
i
o
n

l
i
m
i
t

(
n
g


m
L
-
1
)
R
S
D
%

(
4
)

l
o
w

l
e
v
e
l

h
i
g
h

l
e
v
e
l

R
u
t
i
n

S
.
6

±

u
.
6

y
=

(
u
.
7
S
6

±

u
.
u
u
9
)

x

+

9
.
2
1
1

±

u
.
u
S
8

u
.
9
9
7
4

1
.
1
S

(
u
.
S
8
)

S
.
u

-

8
u
u

1
.
S

S
.
9

6
.
2

N
a
i
i
n
g
i
n

7
.
u

±

u
.
2

y
=

(
u
.
S
u
6

±

u
.
u
u
S
)

x

+

4
.
u
u
2

±

u
.
1
8
2

u
.
9
9
S
9

u
.
S
1

(
u
.
9
7
)

1
7
.
8

-

8
u
u

1
.
8

4
.
S

4
.
S

B
e
s
p
e
i
i
u
i
n

7
.
S

±

u
.
2

y
=

(
u
.
2
7
8

±

u
.
u
u
S
)

x

+

1
6
.
8
9
1

±

u
.
2
8
1

u
.
9
9
S
1

u
.
6
2

(
u
.
8
1
)


1
u
.
1

-

1
u
u
u

S
.
u

S
.
8

4
.
7

Q
u
e
i
c
e
t
i
n

9
.
9

±

u
.
6


y
=

(
u
.
S
u
4

±

u
.
u
u
2
)

x

+

1
1
.
u
7
S

±
u
.
S
7
1

u
.
9
9
8
1

u
.
6
1

(
u
.
6
9
)

1
2
.
2

-

4
u
u
u

S
.
7

2
.
8

S
.
2

N
a
i
i
n
g
e
n
i
n

1
u
.
6

±

u
.
4

y
=
(
u
.
S
S
S

±

u
.
u
u
4
)

x

+

1
1
.
8
S
S

±

u
.
2
2
2

u
.
9
9
6
9

u
.
S
6

(
u
.
9
2
)


6
.
7

-

1
u
u
u

2
.
u

S
.
S

4
.
7

K
a
e
m
p
f
e
i
o
l

1
1
.
6

±

u
.
S

y
=

(
u
.
7
7
9

±

u
.
u
u
7
)

x

+

9
.
9
S
7

±

u
.
2
6
9

u
.
9
9
8
8

u
.
S
4

(
u
.
7
1
)

S
.
S

-

1
u
u
u

1
.
u

S
.
9

2
.
8



1
4
8
T
a
b
l
e

4
.

A
p
p
l
i
c
a
t
i
o
n

o
f

t
h
e

m
e
t
h
o
u

N
a
t
u
r
a
l

J
u
i
c
e

1

N
a
t
u
r
a
l

J
u
i
c
e

1


(
N
a
v
e
l
)

N
a
t
u
r
a
l

J
u
i
c
e

2


(
N
a
v
e
l
)

N
a
t
u
r
a
l

J
u
i
c
e

3


(
C
l
e
m
e
n
t
i
n
a
)

C
o
m
m
e
r
c
i
a
l

J
u
i
c
e

1


C
o
m
m
e
r
c
i
a
l

J
u
i
c
e

2

A
n
a
l
y
t
e
s

C
o
n
c
.
F
o
u
n
d

(
±

S
D
)

(
1
)

R
e
c
o
v
e
r
y

(
%
)

C
o
n
c
.

F
o
u
n
d

(
±

S
D
)

(
1
)

R
e
c
o
v
e
r
y

(
%
)

C
o
n
c
.

F
o
u
n
d

(
±

S
D
)

(
1
)

R
e
c
o
v
e
r
y


(
%
)

C
o
n
c
.

F
o
u
n
d

(
±

S
D
)

(
1
)

R
e
c
o
v
e
r
y


(
%
)

C
o
n
c
.

F
o
u
n
d

(
±

S
D
)

(
1
)

R
e
c
o
v
e
r
y


(
%
)





1
s
t

2
n
u



1
s
t

2
n
u



1
s
t

2
n
u



1
s
t

2
n
u



1
s
t

2
n
u

R
u
t
i
n

n
.
u
.

-

-

n
.
u
.

-

-

n
.
u
.

-

-

1
.
4


(
±

u
.
S
)

9
9
.
9

9
1
.
8

S
.
S


(
±

u
.
6
)

9
S

1
u
S

N
a
i
i
n
g
i
n

8
S
.
8


(
±

u
.
S
)

9
9
.
S

9
1
.
6

6
8
.
8


(
±

u
.
4
)

9
7
.
9

1
u
S

1
u
S


(
±

1
)

1
u
2

9
4
.
S

8
7
u


(
±

2
)

9
7
.
9

1
u
S

2
4
4


(
±

1
)

9
S
.
7

9
S
.
7

B
e
s
p
e
i
i
u
i
n

1
9
4
.
8

(
±

u
.
2
)

1
u
1

9
S
.
6

1
6
S
.
S


(
±

u
.
8
)

8
9
.
9

1
u
1

1
8
8
.
9


(
±

u
.
8
)

9
6
.
9

1
u
S

7
6
u

(
±

2
)

8
9
.
9

1
u
1

1
7
2


(
±

1
)

8
6
.
9

1
u
6

Q
u
e
i
c
e
t
i
n

S
6
u


(
±

1
)

9
8
.
S

9
2
.
8

9
u


(
±

1
)

9
1
.
6

9
4
.
4

1
1
6
.
8


(
±

u
.
1
)

1
u
4

8
9
.
9

2
4
S
.
u


(
±

u
.
8
)

9
1
.
6

9
4
.
4

9
6


(
±

2
)

1
u
7

9
u
.
4

N
a
i
i
n
g
e
n
i
n

2
4
8


(
±

2
)

1
u
1

9
6
.
8

6
S


(
±

1
)

1
u
S

9
7
.
8

1
8
6
.
S


(
±

u
.
2
)

1
u
S

9
8
.
9

S
S
u


(
±

S
)

1
u
S

9
7
.
8

4
9


(
±

1
)

1
u
4

9
4
.
6

K
a
e
m
p
f
e
i
o
l

n
.
u
.

-

-

n
.
u
.

-

-

n
.
u
.

-

-

2
6


(
±

2
)

1
u
7

1
u
6

n
.
u
.

-

-


(
1
)

C
o
n
c
e
n
t
r
a
c
i
o
n

i
n

µ
g

m
L
-
1

(
S
D
=

s
t
a
n
d
a
r
d

d
e
v
i
a
t
i
o
n
)
.

(
2
)

R
e
c
o
v
e
r
i
e
s

a
f
t
e
r

a
d
d
i
t
i
o
n

o
f

2
0
.
0

a
n
d

1
0
0
.
0

n
g

m
L
-
1
,

f
i
r
s
t

a
n
d

s
e
c
o
n
d

a
d
d
i
t
i
o
n
,

r
e
s
p
e
c
t
i
v
e
l
y
.

Innovaciones en la determinación cromatográfica de antioxidantes en alimentos

149
3. Conclusions

This aiticle shows the usefulness of a new LC post-column
fluoiescent system foi the uiiect ueteimination of flavonoius in oiange
juices. The use of long-wavelength measuiements to obtain analytical
signals avoius the inteifeience of potential signals fiom the sample matiix.
Also, the low uetection limits ieacheu allow the use of a high uilution
factoi, avoiuing sample tieatment anu pieconcentiation piocesses. These
uetection limits aie, in geneial, lowei than those uesciibeu using
photometiic uetection. Foi instance, the uetection limits iepoiteu in some
of these methous |1u, 12, 1Sj foi naiingin, queicetin, hespeiiuin anu iutin
aie in the iange u.uS-1.2S µg mL-1, u.1-2.S µg mL-1, u.S-1.u µg mL-1 anu
u.u2-u.u4 µg mL-1, iespectively. A potential limitation of the methou is the
ielative low flow-iate selecteu using a monolithic column, which was
iequiieu to make compatible the sepaiation, post-column ueiivatization
anu fluoiescent uetection piocesses.

Acknowledgements

Authois giatefully acknowleuge financial suppoit fiom the Spanish NICINN
(Ninisteiio ue Ciencia e Innovacion) (uiant No. CTQ2uu6-uS26S¡BQ0) anu
the }unta ue Anualucia (uiant No. P06-FQN-u1SS6).
















Capítulo 2

150
References

|1j uomez-Bens, A,; Aguilai-Caballos, N.P.; Tienus Anal. Chem. 2uu4, 2S, 127-
1S6.
|2j Sánchez-Naitinez, N. L.; Aguilai-Caballos, N.P.; uomez-Bens, A.; }. Phaim.
Biomeu. Anal. 2uu4, S4, 1u21-1u27.
|Sj Nanzano-Ayala, B.R.; Feinánuez-Romeio, }.N.; uomez-Bens, A.; Anal.
Chim. Acta. 2uu9, 6S2, 1u9-114.
|4j Sánchez-Naitinez, N.L.; Aguilai-Caballos, N.P.; uomez-Bens, A.; Talanta,
2uu9, 78, SuS-Su9.
|Sj Zheng, B.; Chen, X. L.; Zhu, C. Q.; Li, B. B.; Chen, Q. Y.; Xu, }.u.; Niciochem. }.
2uuu, 64, 26S-269.
|6j Stiohsahl, C. N.; Bu, B.; Nillei, B. L.; Kiauss, T. B.; Talanta, 2uuS, 67, 479-
48S.
|7j Nolnái-Peil, I.; Füzfai, Z.; }. Chiomatogi. A, 2uuS, 1u7S, 2u1-227.
|8j }ustesen, 0.; Knuthsen, P.; Leth, T.; }. Chiomatogi. A, 2uuS, 799, 1u1-11u.
|9j Nouly, P.; uayuou, E. N.; Auffiay, A.; }. Chiomatogi. A, 1998, 8uu, 171-179.
|1uj Belajová, E.; Suhaj, N.; Foou Chem. 2uu4, 86, SS9-S4S.
|11j Abau-uaicia, B.; Beiiueta, L. A.; Lopez-Náiquez, B. N.; Ciespo-Feiiei, I.;
uallo, B.; vicente, F.; }. Chiomatogi. A, 2uu7, 11S4, 87-96.
|12j Caieii, N.; Elviii, L.; Nangia, A.; Nusci, N.; }. Chiomatogi. A, 2uuu, 881,
449-46u.
|1Sj Bugo, P.; Piesti, N. L.; 0hman, N.; Fazzio, A.; Bugo, u.; Nonuello, L.; }. Sep.
Sci. 2uuS, 28, 1149-11S6.
|14j Anuieu-Navaiio, A.; Feinánuez-Romeio, }. N.; uomez-Bens, A.; }. Sep. Sci.
2u1u, SS, Su9-S1S.
|1Sj Nata-Bilbao, N. L.; Anuiés-Lacueva, C.; }áuiegui, 0.; Lamuela-Raventos, R.
N.; Foou. Chem. 2uu7, 1u1, 1742-1747.
|16j uawanui, v. B.; uuha, S. N.; Piiyauaisini, K. I.; Nohan, N.; }. Colloiu.
Inteiface Sci. 2uu1, 242, 22u-229.
|17j Liu, W.; uuo, R.; }. Colloiu Inteiface Sci. 2uu6, Su2, 62S-6S2.
|18j Liu, R. T.; Yang, }. u.; Wu, X.; Bua, S.; Sun, C. X.; Spectiochim. Acta Pait A,
2uu1, S7, 2S61-2S66.
|19j Buang, X.; Yang, }.; Zhang, W.; Zhang, Z.; An, Z.; }. Chem. Euuc. 1999, 76, 9S-
94.
|2uj NcNaught, A. B.; Wilkinson, A.; I0PAC. Compenuium of Chemical
Teiminology, 2
nu
eu. (the "uolu Book"). Blackwell Scientific Publications,
0xfoiu (1997). XNL on-line coiiecteu veision: http:¡¡golubook.iupac.oig
(2uu6). uoi:1u.1SS1¡golubook.













CAPITULO 3
INNOVACIONES EN LA DETERMINACIÓN NO
CROMATOGRÁFICA DE ANTIOXIDANTES EN
ALIMENTOS





















































Innovaciones en la ueteiminacion no ciomatogiáfica ue antioxiuantes en alimentos

1SS
Este capitulo piesenta las investigaciones que han uauo lugai al
uesaiiollo ue uos nuevos métouos paia la ueteiminacion ue antioxiuantes
alimentaiios meuiante meuiuas uinámicas. Los iesultauos ue estos estuuios
se iecogen en los siguientes aiticulos:

- Beteimination of antioxiuant auuitives in fooustuffs by uiiect
measuiement of golu nanopaiticle foimation using iesonalce
light scatteiing uetection. A. Anuieu-Navaiio, }.N. Féinánuez-
Romeio, A. uomez-Bens, Anal. Chim. Acta, 2u11, 69S, 11-17.
- Beteimination of polyphenolic content in beveiages using
laccase, golu nanopaiticles anu long wavelength fluoiimetiy. A.
Anuieu-Navaiio, }.N. Féinánuez-Romeio, A. uomez-Bens, Anal.
Chim. Acta, enviauo.

En el piimeio ue ellos se utiliza la capaciuau ue los antioxiuantes
paia ieuucii al oio(III) en uisolucion, uanuo lugai a la foimacion ue
nanopaiticulas. Este compoitamiento ha peimitiuo la cuantificacion ue
estos compuestos meuiante meuiuas ue la vaiiacion ue la uispeision ue la
iauiacion ue iesonancia con el tiempo y utilizacion ue la velociuau inicial
uel pioceso como paiámetio analitico. En la intiouuccion ue este tiabajo se
comentan los escasos anteceuentes uesciitos sobie la monitoiizacion ue la
foimacion ue nanopaiticulas ue oio (AuNPs) con fines analiticos. No
obstante, auemás ue su aplicacion analitica, cabe uestacai que este estuuio
pone ue manifiesto poi piimeia vez la posibiliuau ue obtenei AuNPs en
solo unos segunuos meuiante un pioceuimiento muy simple.

El segunuo métouo utiliza la fluoiimetiia ue laiga longituu ue onua
paia ueteiminai polifenoles meuiante el efecto inhibiuoi que causan estos
compuestos en la oxiuacion enzimática uel fluoiofoio veiue ue inuocianina,
en piesencia ue AuNPs caigauas positivamente. Este sistema competitivo
oiigina un peiiouo ue inuuccion que es piopoicional a la concentiacion ue
los analitos, en el que éstos impiuen la oxiuacion uel fluoiofoio, con la
coiiesponuiente péiuiua ue su fluoiescencia. Paia iealizai este estuuio se
eligio la enzima laccasa que, a uifeiencia ue otias oxiuasas, no iequieie el
uso ue ieactivos como el peioxiuo ue hiuiogeno paia actuai como
catalizauoi ya que ieuuce al oxigeno uisuelto, tiansfoimánuolo en agua,
mientias se oxiua el sustiato. Aunque se han uesciito uiveisos sustiatos
coloieauos ue la laccasa |1j, es la piimeia vez que se utiliza la fluoiimetiia
ue laiga longituu ue onua paia la ueteiminacion ue polifenoles. 0tio
aspecto noveuoso uel métouo piopuesto es el uso ue AuNPs paia mejoiai
Capitulo S

1S4
los limites ue ueteccion obteniuos, uebiuo a la inteiaccion ue las NPs con la
enzima.

Entie los objetivos planteauos paia el uesaiiollo ue estos métouos
se encuentia la búsqueua ue nuevas aplicaciones analiticas ue las AuNPs,
cuyo uso ha teniuo un gian impacto en los últimos años en la Quimica
Analitica. Las especiales piopieuaues opticas y eléctiicas que piesentan
estas NPs han siuo utilizauas en numeiosos métouos ueteiminativos, tales
como los basauos en el uesaiiollo ue biosensoies o ue nuevos maicauoies
en inmunoensayo, y en estuuios ue hibiiuacion ue áciuos nucleicos |2j.
También se han uesciito uiveisas aplicaciones ue las AuNPs en piocesos ue
sepaiacion y pieconcentiacion utilizanuo técnicas ciomatogiáficas y
electiofoiéticas |Sj. Sin embaigo, su uso con fines ueteiminativos paia la
ueteccion uiiecta ue los analitos meuiante el seguimiento ue la sintesis ue
las AuNPs, o su utilizacion como inhibiuoies ue la activiuau enzimática,
como se uesciibe en este capitulo, ha siuo muy iestiingiuo.



Bibliografía

|1j Kulys, }.; Biatkovskaja, I.; Talanta, 2uu7, 72, S26-SS1.
|2j uomez-Bens, A.; Feinánuez-Romeio, }. N.; Aguilai-Caballos, N. P.
Tienus. Anal. Chem., 2uu8, 27, S94-4u6.
|Sj Wu, C. S.; Liu, F. K.; Ko, F. B.; Anal. Bioanal. Chem., 2u11, S99, 1uS-
118L. Rieux, B. Nieueilänuei, E. veipooite, R. Bischoff, }. Sep. Sci.,
2uuS, 28, 1628-1641.














Innovaciones en la ueteiminacion no ciomatogiáfica ue antioxiuantes en alimentos

1SS



Analytica Chimica Acta 69S (2u11) 11-17

Determination of Antioxidant Additives in Foodstuffs
by Direct Measurement of Gold Nanoparticle
Formation using Resonance Light Scattering Detection


A. Andreu-Navarro, J.M. Fernández-Romero, A. Gómez-Hens
Department of Analytical Chemistry, Faculty of Sciences, University of Córdoba,
“Marie Curie Annex” Building. Campus of Rabanales, E-14071 Córdoba, Spain


The capability of antioxiuant compounus to ieuuce golu(III) to golu
nanopaiticles has been kinetically stuuieu in the piesence of
cetyltiimethylammonium biomiue using stoppeu-flow mixing technique
anu iesonance light scatteiing as uetection system. This stuuy has given
iise to a simple anu iapiu methou foi the ueteimination of seveial synthetic
anu natuial antioxiuants useu as auuitives in fooustuff samples. The
foimation of AuNPs was monitoieu by measuiing the initial ieaction-iate of
the system in about S s, using an integiation time of u.1 s. Bynamic ianges
of the calibiation giaphs anu uetection limits, obtaineu with stanuaiu
solutions of the analytes, weie (µmol L
-1
): gallic aciu (u.u4 - u.S9, u.u1),
piopyl gallate (u.u4 - 1.41, u.u1), octyl gallate (u.uS - u.SS, u.u8), uouecyl
gallate (u.u2 - u.Su, u.uu7), butylateu hyuioxyanisol (u.u7 - u.S9, u.uu9),
butylateu hyuioxytoluene (u.u4 - u.S2, u.u1), ascoibic aciu (u.11 - 1.72,
u.uS) anu souium citiate (u.u7 - 1.29, u.u2). The iegiession coefficients
weie highei than u.994 in all instances. The piecision of the methou,
expiesseu as RSB%, was establisheu at two concentiation levels of each
analyte, with values ianging between u.6 anu 4.8 %. The piactical
Capitulo S

1S6
usefulness of the uevelopeu methou was uemonstiateu by the
ueteimination of seveial antioxiuant auuitives in fooustuff samples, which
weie extiacteu, appiopiiately uiluteu anu assayeu, obtaining iecoveiies
between 9S.4 anu 99.S %. The iesults obtaineu weie valiuateu using two
iefeience methous.

































Innovaciones en la ueteiminacion no ciomatogiáfica ue antioxiuantes en alimentos

1S7
Introduction

Natuial anu synthetic antioxiuants aie a gioup of compounus useu
as auuitives to pievent oi ietaiu oxiuation ieactions in foou piouucts.
Theie is a tienu to limit the use of synthetic antioxiuants, owing to theii
potential toxic effects, but they aie still founu in a ielatively wiue iange of
fooustuffs, although theii use is subject to veiy stiict safety iegulations |1-
6j. Phenolic compounus such as piopyl (Pu), octyl (0u) anu uouecyl (Bu)
esteis of gallic aciu (uA), butylateu hyuioxyanisole (BBA) anu butylateu
hyuioxytoluene (BBT) aie the main synthetic antioxiuants still authoiizeu,
although they aie being ieplaceu by natuial antioxiuants, such as ascoibic
(AA) anu citiic (CA) acius. CA is also useu as a foou auuitive foi othei
puiposes, such as aciuifiei anu flavouiing agent.

The ueteimination of synthetic antioxiuants in foou samples is
mainly caiiieu out by using ieveise-phase liquiu chiomatogiaphy (LC)
with photometiy oi mass spectiometiy (NS) as uetection systems |7, 8j.
Nethous baseu on gas chiomatogiaphy (uC), with flame ionization uetectoi
(FIB) oi NS, anu capillaiy electiophoiesis (CE) with photometiic oi
electiochemical uetection have been also uesciibeu |7j. The limits of
uetection (L0Bs) iepoiteu foi most of these methous aie at the level of
µmol L
-1
. These methous aie veiy useful foi the iuentification of the
analytes, but they aie time-consuming foi scieening puiposes.

The use of golu nanopaiticles (AuNPs) as analytical ieagents has
alloweu the uevelopment of veiy sensitive methous baseu on theii special
optical anu electiochemical piopeities. Foi instance, they have been useu
as labels in immunoassays anu hybiiuization assays anu as nanoscaffolus to
uevelop chemical sensois |9j. These methous iequiie the pievious
synthesis of the NPs, using geneially tetiachloioauiic aciu anu a ieuucing
ieagent, usually citiate |1u,11j. 0thei ieagents such as cysteine |12j,
ascoibic aciu |1u, 1S, 14j, souium boiohyuiiue |1S, 16j anu gallic aciu |17,
18j have also shown theii usefulness foi this puipose. The expeiimental
conuitions chosen to obtain AuNPs aie ciitical factois that affect the size,
shape anu potential aggiegation of the NPs anu, consequently, theii
piopeities. Some of these methous |14, 1Sj involve the use of
cetyltiimethylammonium biomiue (CTAB) to favoui the synthesis of the
NPs. The foimation of ion paiis between AuCl
4
-
anu cationic suifactants
Capitulo S

1S8
piioi to the foimation of AuNPs was uesciibeu |19, 2uj. Also, the
electiochemical foimation of AuNPs in the piesence of CTAB has been
iecently iepoiteu |21j.

An alteinative appioach to the use of AuNPs as analytical ieagents
is the uiiect foimation of these NPs by using AuCl
4
-
as ieagent, which ieacts
with an analyte that shows ieuucing piopeities. The measuiement of a
piopeity of the AuNPs foimeu allows the uiiect quantification of the
analyte. An auvantage of this appioach is that the contiol of the size anu
shape of the NPs foimeu anu theii stabilization to avoiu theii aggiegation
aie not iequiieu, pioviueu that the expeiimental vaiiables aie suitably
contiolleu. This appioach has been applieu to the ueteimination of
thioglycolic aciu in cosmetic samples using a flow-injection system anu
measuiing the absoibance of the iesonance plasmon banu of the AuNPs
obtaineu |22j. This optical piopeity has been also useu to ueteimine
hyuioquinone in phaimaceutical piepaiations anu catechol anu pyiogallol
in watei samples |2Sj, using a batch foimat. Resonance light scatteiing
(RLS) measuiements have been uesciibeu foi the ueteimination of
hyuiogen peioxiue |24j using AuNPs seeus, which aie enlaigeu in the
piesence of AuCl
4
-
, CTAB anu the analyte, incieasing the RLS signal. This
appioach has been extenueu to the ueteimination of glucose using glucose
oxiuase to obtain hyuiogen peioxiue, but the methou has not been applieu
to the analysis of ieal samples anu it is necessaiy to wait foi 1S min to
obtain each measuiement.

This aiticle uesciibes a methou foi the fast anu automatic
ueteimination of seveial antioxiuant auuitives (Figure 1) in fooustuffs
baseu on the capability of these compounus to ieuuce AuCl
4
-
to AuNPs in
the piesence of CTAB (Figure 1). The foimation of AuNPs is followeu by
monitoiing the vaiiation of the RLS signal with time, using stoppeu-flow
mixing technique, which allows the measuiement of the initial iate of the
system. The L0Bs obtaineu, at the level of nmol L
-1
, aie lowei than those
uesciibeu using absoibance measuiements |22, 2Sj. Although the pioposeu
appioach uoes not uistinguish among the uiffeient antioxiuants, it can be
useu as a veiy sensitive anu fast scieening methou to uetect the piesence of
these compounus in fooustuffs.


Innovaciones en la ueteiminacion no ciomatogiáfica ue antioxiuantes en alimentos

1S9

1. Experimental

1.1. Appaiatus anu Instiuments

A Caiy Eclipse vaiian spectiofluoiimetei (Walnut Cieek, CA, 0SA)
was useu. The instiument was fuinisheu with an RX-2uuu Rapiu Kinetic
System Stoppeu-Flow mixing accessoiy fiom Applieu Photophysics
(Leatheiheau, 0K), which was fitteu with an obseivation cell of 1u x 2 mm
length, 6u µL of innei volume anu contiolleu by the associateu electionics,
the computei anu a pneumatic syiinge uiive system. The tempeiatuie of
the solution was kept constant at the selecteu tempeiatuie by ciiculating
watei fiom a Baake BC1 theimostatic bath (TheimoElection, Kailsiuhe,
ueimany).

1.2. Reagents

All chemicals useu weie of analytical ieagent giaue. Stock solutions
(1uu mg mL
-1
) of the analytes (all supplieu by Sigma) weie piepaieu as
follows: gallic aciu (uA), piopyl gallate (Pu), octyl gallate (0u), uouecyl
gallate (Bu), butylateu hyuioxyanisol (BBA), butylateu hyuioxytoluene
(BBT) weie uissolveu in absolute ethanol. Ascoibic aciu (AA) anu souium
citiate (SC) weie piepaieu using ueionizeu watei puiifieu with a Nilli-Q
system (Nillipoie, Beufoiu, Na, 0SA). Stock anu inteimeuiate solutions
weie stoieu at 4 ºC in the uaik anu weie stable foi at least two weeks.
Woiking stanuaiu solutions weie piepaieu fiom inteimeuiate solutions by
theii uilution in uistilleu watei. Tetiachloioauiic aciu tiihyuiate was also
puichaseu fiom Sigma. 0thei ieagents useu weie souium chloiiue (Sigma),
acetic aciu (Neick), souium uouecyl sulphate (SBS, Sigma), Tiiton X-1uu
(Sigma) anu cetyltiimetylammonium biomiue (CTAB, Sigma).




F
i
g
u
r
e

1
.

C
h
e
m
i
c
a
l

s
t
i
u
c
t
u
i
e
s

o
f

t
h
e

a
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t
i
o
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i
u
a
n
t

c
o
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o
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u
s

a
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e
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.

T
h
e

s
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i
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i
e

o
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i
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a
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o

s
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o
w
n
.
Innovaciones en la ueteiminacion no ciomatogiáfica ue antioxiuantes en alimentos

161
1.S. Nanifolu anu Pioceuuie

Figure 2 shows the stoppeu-flow mixing uevice useu foi the iapiu
mixtuie of the ieactants. A solution containing AuCl
4
-
(1.u mmol L
-1
),
piepaieu in 2u mmol L
-1
acetic¡acetate buffei, aujusteu to pB = 4, was useu
to fill one of the two uiiving syiinges of the stoppeu-flow mouule. The othei
syiinge was filleu with a pie-mixeu solution containing sample oi stanuaiu
anu CTAB (u.2 mmol L
-1
) uilute in the same buffei solution. The pneumatic
syiinge contiol uevice alloweu the automation of each injection. In each iun,
u.1S mL of each solution was mixeu in the mixing chambei of the stoppeu-
flow mouule anu the vaiiation of the RLS signal with time was monitoieu at
i
ex
= i
em
= S4u nm (Ai = u nm) using a spectiofluoiimetei. Each stanuaiu oi
sample solution was assayeu at least six-times. All measuiements weie
caiiieu out at the selecteu optimal tempeiatuie of 2S ± u.1 °C.






















Figure 2. Scheme of the instiumental system: S1 anu S2, uiiving syiinges
containing BAuCl
4
anu CTAB + antioxiuant sample, iespectively; SS,
stopping syiinge; FB, spectiofluoiimetei; C, obseivation cell; T,
theimostat; PC, computei.


Capitulo S

162
Bata weie acquiieu anu piocesseu using the softwaie incoipoiateu
in the spectiofluoiimetei foi application of the initial-iate kinetic methou.
The initial iate (v
0
) was estimateu by measuiing the slope of the kinetic
cuive in the inteival u - S s, being the integiation time u.1 s. The lineai
calibiation giaph foi each analyte was obtaineu by plotting the uiffeience
obtaineu foi the initial iate in the piesence anu absence of the analyte
veisus the analyte concentiation. The concentiation of the antioxiuant
compounu in the samples was ueteimineu by inteipolation in the
calibiation giaphs.

1.4. Chaiacteiization of the AuNPs

Tiansmission election micioscopy (TEN) images of uiffeient
samples weie obtaineu by uiopping the iesulting AuNPs on a caibon-coateu
coppei giiu anu setting a completely uiieu uiop by vacuum uesiccatoi. The
chaiacteiization of the NPs was peifoimeu by Tiansmission Election
Nicioscopy (TEN), using a CN-1u Philips Nicioscope with u.S x u.S4 nm
iesolution anu equippeu with a uigital megaview III cameia. Coppei giius
(2uuC-FC) coateu with a Foimvai¢ caibon film 2uu mesh, supplieu by
Aname (Nauiiu, Spain), weie useu as suppoit in TEN expeiiments.

1.S. Analysis of Fooustuff Samples

Foui commeicial fooustuffs, incluuing biscuits, uoughnuts,
milkshakes anu soup stock cubes, weie piocesseu to ueteimine theii
coiiesponuing antioxiuant concentiations. The extiaction step was caiiieu
out following the official extiaction pioceuuie uesciibeu elsewheie |2Sj. In
summaiy, 1 g of each sample was exactly weigheu in a tube anu then
extiacteu thiee times using S-mL of a Su% v¡v methanol¡watei solution.
The extiacts weie mixeu foi S min anu centiifugeu at Suuu ipm foi S min.
The uppei solution was collecteu anu uiluteu with an auequate volume of
buffei, in oiuei to fit the lineai iange of the calibiation giaph, anu was
tieateu as uesciibeu above. Each ueteimination was caiiieu out in
tiiplicate, using in each instance the mean of the uata obtaineu fiom at least
six kinetic cuives.



Innovaciones en la ueteiminacion no ciomatogiáfica ue antioxiuantes en alimentos

16S
1.S.1. Refeience Nethous

Each sample was testeu by the A0AC official methou 98S.1S foi the
ueteimination of phenolic antioxiuants in oils, fats anu buttei |26j. Biiefly,
the antioxiuants weie extiacteu in acetonitiile anu the extiact was
concentiate anu ie-uissolveu in 2-piopanol. A volume (1u µL) of this
solution was analyzeu by LC with 0v uetection at 28u nm. This methou
allows the ueteimination of Pu, 0u, Bu, 2,4,S-tiihyuioxybutiiophenone
(TBBP), teit-butylhyuioquinone (TBBQ), noiuihyuioguaiaiacetic aciu
(NBuA), BBA, BBT anu 2, 6-ui teibutyl-4-hyuioxymethylphenol (Ionox
1uu).
The content of AA in the milkshake sample analyzeu was
ueteimineu by anothei iefeience methou baseu on the titiation with N-
biomosuccinimiue (NBS), using souium iouiue anu staich as the inuicatoi
system anu oxalic aciu as stabilizei |27j. Biiefly, the pioceuuie was as
follows: a volume of the sample (1u mL) was centiifugeu anu S mL of the
supeinatant solution weie collecteu anu iisen up to a final volume of 2Su
mL. A volume (2u mL) of this solution was tiansfeiieu to a stanuaiu flask
anu 1.S mL 1u% KI solution, 2 mL 98% acetic aciu solution anu S-4 uiops of
staich solution weie auueu. The titiation was caiiieu out with u.1 mmol L
-1

NBS stanuaiu solution. All titiations weie peifoimeu in tiiplicate.


2. Results and discussion

2.1. Stuuy of the Foimation of AuNPs

Seveial synchionous spectia (Ai = u nm) weie obtaineu to stuuy
the behavioui of the system anu choose the optimum wavelengths foi RLS
measuiements, using uA as the antioxiuant mouel. Figure 3.A uepicts the
synchionous spectia obtaineu foi solutions containing AuCl
4
-
in the
piesence of uA, CTAB, anu both uA anu CTAB. The ieactants weie
pieviously mixeu using stoppeu-flow technique. As can be seen, the
solution containing AuCl
4
-
anu uA uoes not give any scatteiing signal, which
shows that AuNPs aie not foimeu unuei these conuitions. Although uA has
been uesciibeu as ieagent foi the foimation of AuNPs in the absence of
CTAB, it is necessaiy to wait foi at least Su min |17, 18j. The solution
containing AuCl
4
-
anu CTAB shows a ielatively high scatteiing banu, with a
Capitulo S

164
wiue maximum at 4uu-Suu nm, which is asciibeu to the foimation of
insoluble ion paiis between these species |19, 2uj anu, also, to the emission
of the light souice in the spectiofluoiimetei. The synchionous spectium
obtaineu foi uA in the piesence of the mixtuie CTAB-AuCl
4
-
shows also a
shouluei at about S2u-6uu nm that is inuicative of the foimation of AuNPs.
The use of an anionic oi no ionic suifactant such as SBS oi Tiiton X-1uu,
iespectively, alone oi togethei with uA uiu not give any scatteiing signal,
obtaining spectia similai to that obtaineu foi the solution containing AuCl
4
-

anu uA (cuive 1 in Figuie S.A). These iesults show that the foimation of the
ionic paii between AuCl
4
-
anu the cationic suifactant is necessaiy, unuei
the expeiimental conuitions useu, to obtain the NPs. Figure 3.B shows a
TEN image obtaineu foi a solution containing AuCl
4
-
, CTAB anu uA, in
which the foimation of AuNPs can be seen with a wiue size iange, although
most of them iangeu between 1u anu 2u nm.

Figure 3.C shows the kinetic cuives obtaineu in the absence anu in
the piesence of uiffeient uA concentiations using i
ex
= i
em
= S4u nm anu
stoppeu-flow mixing technique. As can be seen, the initial iate can be
measuieu in about S s. The low initial iate of the CTAB- AuCl
4
-
system in the
absence of uA (cuive 1) shows the usefulness of kinetic methouology as it
avoius the ielatively high backgiounu signal (Figure 3.A, cuive 2) that
woulu be obtaineu using equilibiium measuiements. The ieactants weie
uistiibuteu in the syiinges by intiouucing the AuCl
4
-
solution in one of the
syiinges anu using a piemixeu solution containing CTAB anu the analyte to
fill the othei syiinge. It was not possible to use a solution containing the
mixtuie of AuCl
4
-
anu CTAB owing to the foimation of the insoluble ion
paiis. Regaiuing the solution containing AuCl
4
-
anu the analyte, it showeu a
clouuy aspect aftei seveial minutes, which coulu be asciibeu to the slow
ieuuction of Au(III).


F
i
g
u
r
e

3
.

(
A
)

S
y
n
c
h
i
o
n
o
u
s

s
p
e
c
t
i
a

o
b
t
a
i
n
e
u

a
f
t
e
i

s
t
o
p
p
e
u
-
f
l
o
w

m
i
x
i
n
g

o
f

s
o
l
u
t
i
o
n
s

c
o
n
t
a
i
n
i
n
g

A
u
C
l
4
-

i
n

t
h
e

p
i
e
s
e
n
c
e

o
f

u
A

(
1
)
,

C
T
A
B

(
2
)

a
n
u

C
T
A
B

+

u
A

(
S
)

|
u
A
j

=

u
.
1

µ
m
o
l

L
-
1
)
.

(
B
)

T
E
N

i
m
a
g
e

o
b
t
a
i
n
e
u

f
o
i

a

s
o
l
u
t
i
o
n

c
o
n
t
a
i
n
i
n
g

A
u
C
l
4
-

+

C
T
A
B

+

u
A

(
u
.
S

µ
m
o
l

L
-
1
)
.

(
C
)

K
i
n
e
t
i
c

c
u
i
v
e
s

o
b
t
a
i
n
e
u

f
o
i

u
i
f
f
e
i
e
n
t

c
o
n
c
e
n
t
i
a
t
i
o
n
s

o
f

u
A

(
µ
m
o
l

L
-
1
)

=

(
1
)

u
.
u
,

(
2
)

u
.
1
,

(
S
)

u
.
2
,

(
4
)

u
.
S
.


|
A
u
C
l
4
-
j

=

1
.
u

m
m
o
l

L
-
1
;

|
C
T
A
B
j

=

u
.
2

m
m
o
l

L
-
1

(
c
o
n
c
e
n
t
i
a
t
i
o
n
s

a
i
e

i
e
f
e
i
i
e
u

t
o

t
h
e

v
a
l
u
e
s

i
n

t
h
e

s
y
i
i
n
g
e
s
)
.

Capitulo S

166
2.2. 0ptimization of vaiiables

The vaiiables affecting the system weie optimizeu following the
univaiiate methou. All iepoiteu concentiations aie initial concentiations in
each syiinge, which aie twice the final concentiations in the ieaction
mixtuie at time zeio. The analytical paiametei useu was the uiffeience
obtaineu foi the initial iate of the system in the piesence anu absence of
uA. Each kinetic iesult was the aveiage of at least six measuiements. Table
1 summaiizes the vaiiables stuuieu, incluuing the iange assayeu anu the
optimum values chosen.

Table 1. 0ptimization of vaiiables
Type
of variable
Variable
Range
studied
Optimum
value
Instrumental
Excitationwavelength (nm)
Emission wavelength (nm)
Ex¡Em slits (nm)
Powei eneigy (v)
Aveiage time (s)
28u - 8uu
28u - 8uu
2 - 2u
Suu - 7uu
u.u12 - u.1S
S4u
S4u
S¡S
4Su
u.1
Stopped-flow
device
Reaction volume (mL)
Tempeiatuie (°C)
u.1- u.S
1u - Su
u.S
2S
Chemical
pB
|BAc¡Ac
-
j

Buffei (mmol L
-1
)
|AuCl
4
-
j (mmol L
-1
)
|CTABj (mmol L
-1
)
2 - 6
1u - 2uu
u.u - 2.S
u.u - u.S
4.u
2u
1.u

u.2


The initial iate was inuepenuent of the mixing cell volume, which
was assayeu in the iange u.1 - u.S mL, choosing a volume of u.S mL. The
effect of the concentiation of AuCl
4
-
was stuuieu in the inteival u.u - 2.S
mmol L
-1
(Figure 4.A). As can be seen, the optimum concentiation was
close to 1.u mmol L
-1
. The influence of the tempeiatuie on the AuNPs
foimation was stuuieu in the iange 1u - Su °C (Figure 4.B), obtaining the
maximum initial iate at 2S ºC. Figure 4.C shows the pB uepenuence of the
system, which was stuuieu in the iange of 2 - 6. A pB of 4.u was selecteu as
the optimum value, using an acetic aciu¡souium acetate buffei solution to
aujust this pB. The stuuy of the influence of the concentiation of this buffei
Innovaciones en la ueteiminacion no ciomatogiáfica ue antioxiuantes en alimentos

167
on the piocess, assayeu in the iange of 1u - 2uu mmol L
-1
, showeu that a
concentiation of 2u mmol L
-1
concentiation was suitable to aujust the pB.
Seveial souium chloiiue concentiations, in the iange of u-2uu mmol L
-1
,
weie assayeu to evaluate the influence of the ionic stiength on the system,
but the iesults obtaineu showeu that this vaiiable uoes not mouify the
kinetic behavioui of the system.

Finally, the stuuy of the influence of the CTAB concentiation in the
inteival u.u - u.S mmol L
-1
(Figure 4.D) showeu that the optimum
concentiation was u.2 mmol L
-1
. Bighei concentiation values causeu an
inciease of the blank signal, uecieasing the net initial iate coiiesponuing to
uA.



Figure 4. Stuuy of the influence of AuCl
4
-
concentiation (A), tempeiatuie (B), pB
(C), anu CTAB concentiation (B) on the initial iate of the system in the
piesence of u.S µmol L
-1
uA. |AuCl
4
-
j = 1.u mmol L
-1
on Figuies B, C anu B;
tempeiatuie = 2S ºC on Figuies A, C anu B; pB = S on Figuies A anu B,
anu 4 on Figuie B; |CTABj = u.2 mmol L
-1
on Figuies A, B anu C.

Capitulo S

168
2.S. Analytical Featuies

Calibiation giaphs weie iun unuei the optimal expeiimental
conuitions summaiizeu in Table 1, using the initial iate as the analytical
paiametei. Seven stanuaiu solutions containing uiffeient concentiations of
each analyte (uA, Pu, 0u, Bu, BBA, BBT, AA anu SC), coveiing the iange
between u.u1 anu 2 µmol L
-1
, weie assayeu in tiiplicate. Table 2
summaiizes the figuies of meiit of the methou, incluuing the calibiation
equations, lineai ianges, L0Bs anu piecision uata. The L0Bs obtaineu,
calculateu accoiuing to I0PAC iecommenuations |28j iangeu between
u.uu7 µmol L
-1
foi Bu anu u.uS µmol L
-1
foi AA. The piecision, expiesseu as
ielative stanuaiu ueviation (RSB %) was stuuieu at two concentiations of
each analyte, which coiiesponu to the minimal (centioiu point) anu
maximal (close to the limits of quantification) eiiois of the uynamic
calibiation giaphs. The RSB% values, obtaineu foi n = 11 solutions, iangeu
between u.6 anu 4.8 %. The estimateu sampling fiequency unuei the
woiking conuitions was about 8 h
-1
.

2.4. Application of the methou

The pioposeu methou was applieu to the analysis of foui
commeicial fooustuffs, incluuing biscuits, uoughnut, milkshake anu soup
stock cubes, to ueteimine theii coiiesponuing antioxiuant concentiations.
The samples weie analyseu following the sampling pioceuuie uesciibeu in
the expeiimental section. Each sample was extiacteu using the above
mentioneu official extiaction piocess |2Sj anu then auequately uiluteu with
uistilleu watei to match the lineai iange of the calibiation giaph foi each
analyte. The methou was testeu in two ways: 1) by ueteimining the
antioxiuant concentiation in the samples, anu 2) by stuuying the iecoveiy
aftei auuition of two stanuaiu aliquots of the coiiesponuing analyte at two
concentiation levels which coveieu the lineai iange of the calibiation
giaph. The iesults achieveu aie summaiizeu in Table 3, which incluue the
commeicial name, the fooustuff type, the uilution factoi, the concentiations
obtaineu by both iefeience anu pioposeu methous anu the iecoveiy uata
obtaineu at two concentiation levels, which aie uepicteu in biacket. The
antioxiuant content of the sample that containeu two analytes (soup stock
cubes) is expiesseu as Pu concentiation. As can be seen, the iesults
obtaineu by both the pioposeu anu iefeience methous aie veiy similai. The
Innovaciones en la ueteiminacion no ciomatogiáfica ue antioxiuantes en alimentos

169
iecoveiy peicentages obtaineu, calculateu by subtiacting the iesults
obtaineu fiom similai unspikeu samples, iangeu between 9S.4 anu 99.S %.

3. Conclusions

This stuuy shows foi the fiist time the usefulness of kinetic
methouology foi monitoiing the foimation of AuNPs inuuceu by
antioxiuant compounus in the piesence of CTAB anu its analytical
usefulness. This appioach has alloweu the uevelopment of a kinetic methou
foi the ueteimination of uA, Pu, 0u, Bu, BBA, BBT, AA anu SC by measuiing
the vaiiation of RLS signal with time. In contiast to equilibiium methous
involving the piesence of AuCl
4
-
, the use of stoppeu-flow mixing technique
uoes not iequiie an incubation step, obtaining the analytical signal in few
seconus. The iesults obtaineu show that the methou can ieauily be auapteu
as a fast scieening methou to automatic quality contiol of antioxiuant
auuitives in fooustuffs.

Acknowledgements

We giatefully acknowleuge financial suppoit fiom the Spanish
NICINN (Ninisteiio ue Ciencia e Innovacion) (uiant No. CTQ2uu9-
u8621¡BQ0) anu the }unta ue Anualucia (uiant No. P09-FQN-49S).



T
a
b
l
e

2
.

A
n
a
l
y
t
i
c
a
l

f
e
a
t
u
i
e
s

o
f

t
h
e

m
e
t
h
o
u


A
n
a
l
y
t
e

E
q
u
a
t
i
o
n

(
1
)

r
2

σ
y
/
x
(
2
)

L
i
n
e
a
r

r
a
n
g
e

(
µ
m
o
l

L
-
1
)

D
e
t
e
c
t
i
o
n

L
i
m
i
t

(
µ
m
o
l

L
-
1
)


R
S
D
%

(
3
)
l
o
w

l
e
v
e
l

h
i
g
h

l
e
v
e
l

u
a
l
l
i
c

A
c
i
u

(
u
A
)

y
=

(
1
.
1
7

±

u
.
u
4
)

x

+

u
.
u
6

±

u
.
u
u
S

u
.
9
9
7
u

u
.
u
1
S

u
.
u
4

-

u
.
S
9

u
.
u
1

4
.
8

u
.
9

P
i
o
p
y
l

u
a
l
l
a
t
e

(
P
u
)

y
=

(
u
.
4
8

±

u
.
u
1
)

x

+

u
.
u
6
9

±

u
.
u
u
2

u
.
9
9
9
6

u
.
u
u
S

u
.
u
4

-

1
.
4
1

u
.
u
1

6
.
8

S
.
S

0
c
t
y
l

u
a
l
l
a
t
e

(
0
u
)

y
=

(
1
.
4
6

±

u
.
u
6
)

x

-

u
.
u
4
8

±

u
.
u
u
6

u
.
9
9
4
S

u
.
u
1
S

u
.
u
S

-

u
.
S
S

u
.
u
u
8

S
.
S

S
.
S

B
o
u
e
c
y
l

u
a
l
l
a
t
e

(
B
u
)

y
=

(
2
.
S
S

±

u
.
u
S
)

x

+

u
.
1
u
6

±

u
.
u
u
6

u
.
9
9
9
4

u
.
u
u
7

u
.
u
2

-

u
.
S
u

u
.
u
u
7

4
.
2

1
.
7

B
u
t
y
l

B
y
u
i
o
x
y
a
n
i
s
o
l

(
B
B
A
)

y
=

(
1
.
S
8

±

u
.
u
S
)

x

+

u
.
u
S
1

±

u
.
u
u
4

u
.
9
9
8
4

u
.
u
u
7

u
,
u
7

-

u
.
S
9

u
.
u
u
9

1
.
S

S
.
6

B
u
t
y
l

B
y
u
i
o
x
y
t
o
l
u
e
n
e

(
B
B
T
)

y
=

(
2
.
S
9

±

u
.
1
S
)

x

+

u
.
u
2
6

±

u
.
u
1
u

u
.
9
9
4
S

u
.
u
2
S

u
.
u
4

-

u
.
S
2

u
.
u
1

1
.
7

u
.
6

A
s
c
o
i
b
i
c

A
c
i
u

(
A
A
)

y
=

(
u
.
8
2

±

u
.
u
1
)

x

+

u
.
1
1
S

±

u
.
u
u
9

u
.
9
9
9
1

u
.
u
1
6

u
.
1
1

-

1
.
7
2

u
.
u
S

2
.
6

S
.
4

S
o
u
i
u
m

C
i
t
i
a
t
e

(
S
C
)

y
=

(
S
.
6
7

±

u
.
1
2
)

x

+

u
.
u
S
S

±

u
.
u
2
S

u
.
9
9
7
6

u
.
u
2
8

u
.
u
7

-

1
.
2
9


u
.
u
2

2
.
9

1
.
9


(
1
)

y

u
e
n
o
t
e
s

t
h
e

a
n
a
l
y
t
i
c
a
l

s
i
g
n
a
l
,

a
n
u

x

i
s

t
h
e

a
n
a
l
y
t
e

c
o
n
c
e
n
t
i
a
t
i
o
n

(
µ
m
o
l

L
-
1
)
,

(
2
)

i
e
s
i
u
u
a
l

s
t
a
n
u
a
i
u

u
e
v
i
a
t
i
o
n

(
o
i

s
t
a
n
u
a
i
u

e
i
i
o
i

o
f

e
s
t
i
m
a
t
e
)
,

(
S
)


i
e
l
a
t
i
v
e

s
t
a
n
u
a
i
u

u
e
v
i
a
t
i
o
n

v
a
l
u
e
s

a
c
h
i
e
v
e
u

a
t

t
w
o

c
o
n
c
e
n
t
i
a
t
i
o
n

l
e
v
e
l
s

o
f

e
a
c
h

a
n
a
l
y
t
e
,

c
o
i
i
e
s
p
o
n
u
i
n
g

t
o

t
h
e

m
a
x
i
m
a
l

a
n
u

m
i
n
i
m
a
l

e
i
i
o
i

z
o
n
e
s

i
n

t
h
e

c
a
l
i
b
i
a
t
i
o
n

g
i
a
p
h
s

(
f
o
i

m
o
i
e

u
e
t
a
i
l
e
u

s
e
e

t
e
x
t
)
.




T
a
b
l
e

3
.

A
p
p
l
i
c
a
t
i
o
n


o
f

t
h
e

m
e
t
h
o
u

S
a
m
p
l
e

N
º

C
o
m
m
e
r
c
i
a
l

N
a
m
e

F
o
o
d
s
t
u
f
f

T
y
p
e

D
i
l
u
t
i
o
n
F
a
c
t
o
r

A
n
a
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Capitulo S

172
References

|1j Euiopean Pailiament anu Council Biiective N_ 9S¡2¡EC of 2u
Febiuaiy 199S on foou auuitives othei than colouis anu
sweeteneis. Biussels. 0fficial }ouinal of the Euiopean
Communities18 Naich 199S. 1S.u8.2uu6.
|2j Regulation (EC) No 1SSS¡2uu8 of the Euiopean pailiament
anu the Council of 16 Becembei 2uu8 on foou auuitives.
0fficial }ouinal of the Euiopean 0nion, S1 Becembei 2uu8,
S1.12.2uu8.
|Sj FBA. 2uu1a. Coue of Feueial Regulations (CFR), 2uu1a. CFR
(Coue of Feueial Regulations), Title 21: Foou anu Biugs.
Chaptei I e Foou anu Biug Auministiation, Bepaitment of
Bealth anu Buman Seivices, Pait 17u e Foou Auuitives, 0ffice
of the Feueial Registei, Washington, BC (Reviseu Apiil, 2uu8).
|4j FBA. 2uu1b. Coue of Feueial Regulations (CFR), 2uu1b. CFR
(Coue of Feueial Regulations), Title 21: Foou anu Biugs.
Chaptei I e Foou anu Biug Auministiation, Bepaitment of
Bealth anu Buman Seivices, Pait 184 e Biiect Foou
Substances affiimeu as geneially iecognizeu as safe (uRAS),
0ffice of the Feueial Registei, Washington, BC (Reviseu Apiil,
2uu8).
|Sj }oint FA0¡WB0 Expeit Committee on Foou Auuitives (}EFCA),
anu Woilu Bealth 0iganization (WB0) (2uuS). Summaiy of
evaluations peifoimeu by the }oint FA0¡WB0 Expeit
Committee on Foou Auuitives (}ECFA 19S6-2uu7).
http:¡¡jecfa.ilsi.oig¡inuex.htm.
|6j CuSFA (199S). Couex geneial stanuaius foi foou auuitives-
pieamble, Couex STAN 192-199S. (Revision 1997, 1999, 2uu1,
2uuS, 2uu4, 2uuS, 2uu6, 2uu7, 2uu8).
http:¡¡www.couexalimentaiius.net¡gsfaonline¡CXS_192e.puf
|7j Anuié, C.; Castanheiia, I.; Ciuz, }. N., Paseiio, P.; Sanches-Silva,
A.; Tienus Foou. Sci. Technol. 2u1u, 21, 229-246.
|8j Xiu-Qin, L.; Chao, }.; Yan-Yan, S.; Nin-Li, Y.; Xiao-uang, C.; Foou
Chem. 2uu9, 11S, 692-7uu.
|9j uomez-Bens, A.; Feinánuez-Romeio, }.N.; Aguilai-Caballos, N.
P.; Tienus Anal. Chem. 2uu8, 27, S94-4u6.
Innovaciones en la ueteiminacion no ciomatogiáfica ue antioxiuantes en alimentos

17S
|1uj Kimling, }.; Naiei, N.; 0kenve, B.; Kotaiuis, v.; Ballot, B.; Plech,
A.; }. Phys. Chem. B, 2uu6, 11u, 1S7uu-1S7u7.
|11j Weng, u.; }ianjun, L.; }ian, Z.; }unwu, Z.; Colloiu Suif. A:
Physicochem. Eng. Aspects, 2u1u, S69, 2SS-2S9.
|12j Zhanfang, N.; Bongliang, B.; Colloius anu Suifaces A:
Physicochem. Eng. Aspects, 2uu8, S17, 229-2SS.
|1Sj Wagnei, }.; Kohlei, }. N.; Nano Lett. 2uuS, S, 68S-691.
|14j Li, Z.; uu, A.; Nicio Nano Lett. 2uu9, 4, 142-147.
|1Sj Lin, }.; Zhou, W.; 0'Connoi, C. }.; Nateiials Lett. 2uu1, 49, 282-
286.
|16j Polte, }.; Eilei, R.; Thünemann, A. F.; Sokolov, S.; Ahnei, T. T.;
Rauemann, K.; Emmeiling, F.; Kiaehneit, R.; ACS Nano, 2u1u,
4, 1u76-1u82.
|17j Wenxing, W.; Qifan, C.; Cha, }.; Bongzhi, Y.; Xingmin, L.;
Shukun, X.; Colloiu Suif. A: Physicochem. Eng. Aspects, 2uu7,
Su1, 7S-79.
|18j Kuan-Wei, B.; Cheng-}u, Y.; Wei-Lung, T.; Biosen. Bioelect.
2u1u, 2S, 984-99u.
|19j Ishizuka, B.; Tano, T.; Toiigoe, K.; Esumi, K.; Neguio, K.;
Colloiu Suif. 1992, 6S, SS7-S4u.

|2uj Leontiuis, E.; Kleitou, K.; Kypiianiuou-Leouiuou, T.; Bekiaii, v.;
Lianos, P.; Langmuii, 2uu2, 18, S6S9-S668.
|21j Abuelmoti, L. u.; Zamboiini, F. P.; Langmuii, 2u1u, S6, 1SS11-
1SS21.
|22j Sieiia-Roueio, N.; Feinánuez-Romeio, }. N.; uomez-Bens, A.;
Niciochem. }. 2u11, 97, 24S-248.
|2Sj Reza-Boimozi-Nezhau, N.; Alimohammaui, N.; Tashkhouiian,
}.; Nehui-Razavian, S.; Spectiochim. Acta. A. 2uu8, 71, 199-
2uS.
|24j Shang, L.; Chen, B.; Beng, L.; Bong, S.; Biosen. Bioelect. 2uu8,
2S, 118u-1184.
|2Sj King, W. P.; }oshep, K. T.; Kissingei, P. T.; }. Assoc. uff. Anal.
Chem. 19u8, 6S, 1S7-142.
|26j Boiwitz, W.; Latimei, u.; A0AC official methou 98S.1S, }. A0AC
Int., 199S, 76, 76S-768.
|27j 0kiei, W.; 0gulesi, N.; Azeez, L.; 0bakachi, v.; 0sunsami, N.;
Nkenchoi, u.; Int. }. Electiochem. Sci. 2uu6, 276-287.
Capitulo S

174
|28j Long, u. L.; Winefoiunei, }. B.; Anal. Chem. 198S, SS, 712A-
724A.










































Innovaciones en la determinación no cromatográfica de antioxidantes en alimentos

175
Determination of polyphenolic content in beverages
using laccase, gold nanoparticles and long
wavelength fluorimetry


Sent to Analytica Chimica Acta, 0ctobei 2u11

A. Andreu-Navarro, J.M. Fernández-Romero, A. Gómez-Hens
Bepaitment of Analytical Chemistiy, Faculty of Sciences, 0niveisity of
Coiuoba, "Naiie Cuiie Annex" Builuing. Campus of Rabanales, E-14u71
Coiuoba, Spain


An enzymatic fluoiimetiic methou foi the ueteimination of
polyphenol compounus in beveiages is uesciibeu, which is baseu on the
tempoial inhibition causeu by these compounus on the oxiuation of the
long wavelength fluoiophoi inuocyanine gieen (i
ex
764 nm, i
em
8u6 nm), in
the piesence of the enzyme laccase anu positive chaigeu golu nanopaiticles
(AuNPs). The oxiuation of the uye gives iise to a fast ueciease in its
fluoiescence, but it is uelayeu by the polyphenol, obtaining a time peiiou
uiiectly piopoitional to its concentiation, which has been useu as the
analytical paiametei. The behavioui of seveial benzeneuiols anu
benzenetiiols in the system anu the mouification of the activity of the
enzyme by its inteiaction with AuNPs have been stuuieu.

The system has been optimizeu using gallic aciu as a polyphenol
mouel, but the uynamic ianges of the calibiation giaphs anu the uetection
limits foi seveial of the polyphenols assayeu weie obtaineu (µmol L
-1
):
gallic aciu (u.1S-S, u.u4), catechol (u.u8-S, u.u1), hyuioquinone (u.uS-2,
u.u1), hyuioxyhyuioquinone (u.u9-S, u.uS), pyiogallol (u.17-S, u.u4). Nost
of the values of the iegiession coefficients weie u.999 anu the piecision of
the methou, expiesseu as RSB% anu checkeu at two concentiation levels of
each analyte, iangeu between 1.8 anu S.6%. The methou has been applieu
to the ueteimination of polyphenol content in seveial fooustuff samples
anu the iesults compaieu with those obtaineu with the stanuaiu Folin-
Ciocalteu methou.
Capítulo 3

176
Introduction

The availability of suitable methous foi the ueteimination of
polyphenolic compounus in foou piouucts is still a veiy active ieseaich
topic, in spite of the high numbei of methous uesciibeu up to uate foi this
puipose |1,2j. This inteiest is justifieu by the lack of stanuaiuiseu methous
anu the wiuely uesciibeu connection between the piesence of these
compounus in foou piouucts anu theii quality piopeities anu health
benefits |Sj.

Nethous foi the ueteimination of polyphenolic compounus in foou
samples can be mainly classifieu in two gioups: 1) methous focuseu to the
inuiviuual iuentification anu quantification of the constituents of a
polyphenol mixtuie in a sample, which involve the use of a sepaiation
technique, such as chiomatogiaphy |1,4j oi capillaiy electiophoiesis |Sj,
anu 2) methous aimeu to ueteimine collectively polyphenols, obtaining the
total polyphenolic content. The choice of each type of methous uepenus on
the objective of the analysis. 0sually, the sepaiation anu iuentification of all
polyphenols piesent in a foou sample is a complex task owing to the high
numbei of potential analytes anu the uiffeiences in theii concentiation
levels.

The seconu type of methous is mainly baseu on the antioxiuant
piopeities of polyphenolic compounus, which aie uue to the ieactivity of
hyuioxyl gioups on the benzene iing. The main limitation of these methous
is that only an appioximate estimation of the polyphenol content is
obtaineu as the iesults aie iefeiieu to a polyphenol mouel, which is chosen
to obtain the calibiation plot, but the antioxiuant activity of these
compounus uepenus on the position anu numbei of hyuioxyl gioups on the
benzene iing. The most iepiesentative methou of this gioup is the
photometiic Folin-Ciocalteu assay, which involves the use of
heteiopolyphosphotungstate-molybuate in an alkaline meuium anu a
iefeience stanuaiu polyphenol, such as gallic aciu (uA) |6j. This methou
can leau to an oveiestimation of total polyphenols uue to its pooi
selectivity as the ieagent ieacts with othei ieuucing non-phenolic
substances, such as ascoibic aciu anu ieuucing sugais, which aie common
foou auuitives oi aie natuially piesent in the samples.
Innovaciones en la determinación no cromatográfica de antioxidantes en alimentos

177
A gioup of methous uevoteu to the ueteimination of total
polyphenol content aie baseu on the use of ampeiometiic biosensois,
which involve the immobilization of phenoloxiuase enzymes, namely
tyiosinase anu laccase, on a suitable electioue. Polyphenols aie
enzymatically oxiuizeu to quinones oi iauicals anu then uetecteu at the
electioue by theii ieuuction cuiients |7j. A limitation of these sensois is
that the piouucts can potentially electiopolymeiize to polyaiomatic
ueiivatives uamaging the electioue suiface anu limiting the life time of the
biosensoi, being necessaiy to piotect the suiface of the biosensoi |7j. Also,
it has been uesciibeu that the ausoiption of oxiuizeu piouucts on the
suiface of the immobilization suppoit often leaus to enzyme inactivation
phenomena |8j. Laccase has been uesciibeu as the most appiopiiate
enzyme foi the uetection of polyphenolic compounus |7,9j, although
biosensois baseu on the immobilization of both laccase anu tyiosinase have
also shown theii usefulness |1u,11j.

Laccases belong to the so-calleu blue-coppei family of oxiuases
chaiacteiizeu by containing foui catalytic coppei atoms. They have founu
seveial biotechnological applications in textile, foou piocessing,
phaimaceutical anu chemical inuustiies |12,1Sj. A featuie of these enzymes
is theii capability to oxiuize uiffeient substiates with the concomitant
ieuuction of oxygen to watei, avoiuing the use of ieagents such as
hyuiogen peioxiue foi theii catalytic action. Seveial laccase-baseu
ampeiometiic biosensois have been uesciibeu foi the ueteimination of
polyphenols in wines |9,1u,1Sj, beeis |7,11j, tea infusions |14j anu fiuit
juices |1Sj using gallic aciu |7, 9-11,1Sj, caffeic aciu |14j oi guaicol |1Sj as
the polyphenol stanuaiu to obtain the calibiation plot. The polyphenol
content values obtaineu with these sensois aie geneially lowei than that
obtaineu by the Folin-Ciocalteu methou.

In this woik, an alteinative methou foi polyphenol ueteimination is
uesciibeu, which is baseu on the tempoial inhibition causeu by these
compounus on the oxiuation of the long wavelength fluoiophoi
inuocyanine gieen (Figure 1), also calleu caiuio gieen, in the piesence of
laccase anu golu nanopaiticles (AuNPs). The behavioui of seveial
polyphenols (Figure 1) on the system has been stuuieu, choosing gallic aciu
as the stanuaiu foi the optimization anu uevelopment of the methou, which
has been applieu to the ueteimination of polyphenol content in seveial
Capítulo 3

178
beveiage samples. The iesults obtaineu weie compaieu with the stanuaiu
Folin-Ciocalteu methou.



















Fig 1. Chemical stiuctuies of inuocyanine gieen anu the polyphenolic compounus
assayeu.

Inuocyanine gieen
Catechol Resoicinol Byuioquinone
Pyiogallol Phloioglucinol Byuioxihyuioquinone uallic aciu
Innovaciones en la determinación no cromatográfica de antioxidantes en alimentos

179
1. Experimental

1.1. Appaiatus anu Instiument

An SLN Aminco (0ibana, IL) AB2 luminescence spectiometei
pioviueu with a 1Su W continuous xenon lamp anu a 7 W pulseu xenon
lamp was useu. The instiument was fuinisheu with an RX-2uuu Rapiu
Kinetic System Stoppeu-Flow mixing accessoiy fiom Applieu Photophysics
(Leatheiheau, 0K), which was fitteu with an obseivation cell of 1u x 2 mm
length, 6u µL of innei volume anu contiolleu by the associateu electionics,
the computei anu a pneumatic syiinge uiive system. The tempeiatuie of
the solution was kept constant at 2uºC by ciiculating watei fiom a Baake
BC1 theimostatic bath (TheimoElection, Kailsiuhe, ueimany).

1.2. Reagents

All chemicals useu weie of analytical ieagent giaue. 4-
Bimethylaminopyiiuine, tetiaoctylammonium biomiue, BAuCl
4
, NaBB
4
anu
Folin-Ciocalteu ieagent weie obtaineu fiom Sigma. Stock solutions (1u
mmol L
-1
) of the analytes weie piepaieu as follows: gallic aciu (uA) (Sigma)
was uissolveu in the minimum amount of ethanol; catechol (CA) (Sigma),
hyuioquinone (BY) (Aluiich), hyuioxyhyuioquinone (BB) (Aluiich),
phloioglucinol (Pu) (Sigma), pyiogallol (PY) (Sigma-Auiich) anu iesoicinol
(RE) (Sigma-Aluiich) weie piepaieu in watei. Stock anu inteimeuiate
solutions weie stoieu at 4 ºC in the uaik anu weie stable foi at least one
week, except foi PY which hau to be piepaieu uaily. Woiking stanuaiu
solutions (S anu 1uu µmol L
-1
) weie piepaieu fiom inteimeuiate solutions
by theii uilution in watei. A stock solution (1Su u.a.¡mL) of laccase (fiom
Trametes versicolor) (Sigma) was piepaieu uaily in
tiis(hyuioxymethyl)aminomethane (Tiis) buffei solution at pB 7,S. 0ne
unit of laccase is uefineu as the amount of enzyme that tiansfoims 1 µmol
of catechol pei minute at pB 4.S anu 2SºC. Positive chaigeu AuNPs weie
synthesizeu |16j anu stoieu at 4 ºC in the uaik anu weie stable foi at least
thiee weeks. A stock solution (1Su µmol L
-1
) of inuocyanine gieen (Sigma)
was piepaieu uaily by its uissolution in watei. All aqueous solutions weie
piepaieu using watei puiifieu with a Nilli-Q system (Nillipoie, Beufoiu,
Na, 0SA).

Capítulo 3

180
1.S. Nanifolu anu pioceuuie

A stoppeu-flow mixing uevice was useu foi the iapiu anu automatic
mixtuie of the ieactants, which is shown in Figure 2. A pie-mixeu solution
containing laccase (u.1 u.a. mL
-1
), positive chaigeu AuNPs (S9,4 µmol L
-1
)
anu 2S mmol L
-1
Tiis buffei solution (pB = 7.S), which was let stanu foi at
least two houis, was useu to fill one of the two uiiving syiinges of the
stoppeu-flow mouule. The seconu syiinge was filleu with a pie-mixeu
solution containing sample oi stanuaiu anu inuocyanine gieen (S.2 µmol
L
-1
) solutions, piepaieu in the same buffei solution. The pneumatic syiinge
contiol uevice alloweu the automation of each injection. In each iun, u.1S
mL of each solution was mixeu in the mixing chambei of the stoppeu-flow
mouule anu the vaiiation of the fluoiescence signal of inuocyanine gieen
with time was monitoieu at i
ex
= 764 anu i
em
= 8u6 nm using a
spectiofluoiimetei. Each stanuaiu oi sample solution was assayeu at least
foui-times. All measuiements weie caiiieu out at the selecteu optimal
tempeiatuie of 2u ± u.1 °C.
















Figure 2. Scheme of the instiumental system: S1 anu S2, uiiving syiinges
containing laccase + AuNPs anu inuocyanine gieen + polyphenolic
compounu, iespectively; SS, stopping syiinge; FB, spectiofluoiimetei;
C, obseivation cell; T, theimostat; PC, computei.

Innovaciones en la determinación no cromatográfica de antioxidantes en alimentos

181
Bata weie acquiieu anu piocesseu using the softwaie incoipoiateu
in the spectiofluoiimetei foi tieatment of kinetic measuiements. The
analytical paiametei useu to quantify the analyte was the uiffeience in the
time necessaiy, expiesseu in seconus, to obseive a ueciease on the
fluoiescence signal in the piesence anu absence of the analyte, as Figure 3
shows. The concentiation of phenolic compounus in the samples was
ueteimineu by inteipolation in the calibiation giaph obtaineu foi uA,
which was chosen as the analyte mouel.

1.4. Synthesis of negative anu positive chaigeu golu nanopaiticles

Negative chaigeu AuNPs weie synthesizeu by the methou iepoiteu
by Tuikevich |17j. Biiefly, S mg of tetiachloioauiic aciu tiihyuiate was
uissolveu in 47,S mL of ueionizeu watei in a iounu-bottom flask anu
tiisouium citiate uehyuiate (1.7 mN) was auueu uiop wise in two minutes
anu then was vigoious stiiieu unuei ieflux at 1uu ºC foi Su min. The final
aqueous solution, containing appioximately u.2S mN of AuNps, was kept
stable in iefiigeiatoi at 4 ºC until use. The size of these AuNPs iangeu fiom
2u to Su nm.

Positive chaigeu AuNPs weie synthesizeu by the methou iepoiteu
in the liteiatuie |16j as follows: a Su mN aqueous tetiachloioauiic aciu
was auueu to a 2S mN solution of tetiaoctylammonium biomiue in toluene
(8u mL). Then a u.4 N solution of fieshly piepaieu NaBB
4
was auueu to the
stiiieu mixtuie, which causeu an immeuiate ieuuction to occui. Aftei Su
min, the two phases weie sepaiateu anu the toluene phase was
subsequently washeu with u.1 N B
2
S0
4
, u.1 N Na0B, anu watei (thiee
times), anu then uiieu ovei anhyuious Na
2
S0
4
. Aftei that, u.1N 4-
uimethylaminopyiiuine (BNAP) solution was auueu to the toluene phase.
Biiect phase tiansfei acioss the oiganic¡aqueous bounuaiy was completeu
within 1h. The size of these AuNPs was fiom S to 1S nm.

1.S. Analysis of fooustuff samples

Commeicial juice anu tea samples weie piocesseu to ueteimine
theii coiiesponuing polyphenol content. }uice samples only iequiieu theii
uiiect uilution with uistilleu watei. Foi tea samples, an infusion of 2,S g tea
bag was piepaieu in Su mL of hot uistilleu watei. The extiacts weie stoieu
Capítulo 3

182
at 4 ºC in the uaik. The solutions weie uiluteu in oiuei to fit the lineai
iange of the calibiation giaph, anu weie tieateu as uesciibeu above. Each
ueteimination was caiiieu out in tiiplicate, using in each instance the mean
of the uata obtaineu fiom at least foui measuiements.

1.S.1. Refeience methou

Each sample was testeu by the Folin-Ciocalteu methou |6j which is
the A0AC official methou foi the ueteimination of total polyphenolic
compounus. Biiefly, the pioceuuie was as follows: The analyte stanuaiu oi
the sample solution was mixeu with u.2S mL of the Folin Ciocalteu ieagent
anu the mixtuie was let to ieact foi a few minutes. Aftei, 1 mL of Na
2
C0
S

satuiateu solution was auueu anu the mixtuie was iisen up to a final
volume of S mL. Aftei 2 h, the absoibance at 76S nm was measuieu. The
lineai calibiation giaph foi uA as the analyte mouel was obtaineu by
plotting the uiffeience obtaineu foi the absoibance values in the piesence
anu absence of the analyte veisus the analyte concentiation. The
polyphenol content in the samples was ueteimineu by inteipolation in the
calibiation giaph.

2. Results and discussion

2.1. Stuuy of the chemical system

The chemical system useu to uevelop the pioposeu methou is baseu
in the capacity of polyphenolic compounus to uelay the oxiuation of the
long wavelength fluoiophoi inuocyanine gieen in the piesence of laccase.
This fluoiophoi was chosen with the aim of establishing a fluoiimetiic
methou in which the potential backgiounu signals fiom the sample matiix
aie avoiueu, as they can appeai at lowei wavelengths. As can be seen in
Figure 3, when the fluoiophoi is mixeu with laccase, its fluoiescence
shows a fast ueciease, which can be asciibeu to the catalytic effect of the
enzyme on the oxiuation of the uye by uissolveu oxygen. Bowevei, this
effect is uelayeu in the piesence of a polyphenol compounu, such as uA,
obtaining an appaient inuuction peiiou that is uiiectly piopoitional to the
concentiation of the polyphenol. These iesults show the sequential
catalytic effect of the enzyme, which acts ovei the fluoiophoi when the
polyphenol has been oxiuizeu. Figure 3 also shows that the slope of the
Innovaciones en la determinación no cromatográfica de antioxidantes en alimentos

183
kinetic cuives obtaineu in the absence anu in the piesence of the
polyphenol has piactically the same value, infeiiing that the kinetics of the
oxiuation of the uye aie not affecteu by the oxiuation piouuct of the fiist
ieaction. It has been uesciibeu that AuNPs can ueciease the activity of
lacasse in a homogeneous meuium owing to theii inteiaction |18j. With the
aim of stuuying the potential effect of these NPs on the system, seveial
assays weie caiiieu out using negative anu positive chaigeu AuNPs. The
fiist type of NPs uiu not mouify the kinetics of the system, but an inciease
on the inuuction time was founu in the piesence of positive chaigeu AuNPs,
as Figure 3 shows. This effect can be asciibeu to a ueciease in the
enzymatic activity of laccase uue to its electiostatic inteiaction with the
NPs, as the assays weie caiiieu out at pB 7.S, in which the enzyme is
negatively chaigeu (isoelectiic point = S.S) |19j. The stuuy of the time
necessaiy to attain a suitable laccase-NPs inteiaction showeu that the
iesults weie satisfactoiy anu iepiouucible when the ieagent mixtuie was
piepaieu at least two houis befoie its use.

The behavioui of seveial ui- (CA, BY, RE) anu tii-phenols (BB, Pu,
PY, uA) on the system was stuuieu with the aim of checking its potential
application to the ueteimination of polyphenols. The iesults obtaineu
showeu that the compounus having the hyuioxyl gioups in position oito oi
paia gave a similai iesponse, as uesciibeu below, but those in position
meta, such as RE anu Pu, gave a lowei iesponse. Also, it was founu that the
piesence of the caiboxylic gioup in uA hau only a slight effect on the
kinetics of the system as the iesponse obtaineu was similai to that obtaineu
foi PY.











Capítulo 3

184

0 200 400 600 800 1000
0,12
0,14
0,16
0,18
0,20
0,22
F
l
u
o
r
e
s
c
e
n
c
e

i
n
t
e
n
s
i
t
y
,

a
.
u
.
Time, s
(1) (3) (2) (4)
FIGURE 3

Figure 3. Kinetic cuives obtaineu foi the laccase-inuocyanine gieen system alone
(1) anu in the piesence of AuNPs (2), uA (S) anu AuNPs + uA (4).
|laccasej = u.1 u.a. mL
-1
, |inuocyanine gieenj = S.2 µmol L
-1
, |uAj = 2
µmol L
-1
, tempeiatuie = 2u ºC, pB = 7.S anu |Tiisj = 2S mmol L
-1
.



2.2. 0ptimization of vaiiables

The vaiiables affecting the system weie optimizeu following the
univaiiate methou. All iepoiteu concentiations aie initial concentiations in
each syiinge, which aie twice the final concentiations in the ieaction
mixtuie at time zeio. As inuicateu above, the analytical paiametei useu was
the uiffeience in the time necessaiy to obseive a ueciease in the
fluoiescence of inuocyanine gieen in the piesence anu absence of uA,
which was calleu inuuction time anu expiesseu in seconus. Each iesult was
the aveiage of at least foui measuiements. Table 1 summaiizes the
Innovaciones en la determinación no cromatográfica de antioxidantes en alimentos

185
vaiiables stuuieu, incluuing the iange assayeu anu the optimum values
chosen.

The mixing cell volume chosen was u.S ml, as this vaiiable hau not
any influence on the system in the iange u.1 - u.S mL. The pB uepenuence
of the system was stuuieu in the iange of 6 - 8.S because the inuocyanine
gieen solution was no stable at pB lowei than 6.u, showing a ueciease in its
fluoiescence intensity, wheieas the laccase solution was unstable at pB
above 8.S, as its catalytic effect thiough the ueciease of the uye
fluoiescence was not obseiveu. A pB 7.S was selecteu as the optimum
value, using a Tiis buffei solution aujusteu at this pB value. The stuuy of
the influence of the concentiation of this buffei on the piocess, assayeu in
the iange of 1u - 2uu mmol L
-1
, showeu that a 2S mmol L
-1
concentiation
was suitable to aujust the pB. Seveial souium chloiiue concentiations, in
the iange of u-2uu mmol L
-1
, weie assayeu to evaluate the influence of the
ionic stiength on the system, but the iesults obtaineu showeu that this
vaiiable uiu not mouify the behavioui of the system. The effect of the
concentiation of positive chaigeu AuNPs on the system was stuuieu in the
inteival u.u - S2.4 µmol L
-1
(Figure 4.A), obtaining an optimum
concentiation close to 4u µmol L
-1
. Laccase activity was a ciitical vaiiable,
as can be seen in Figure 4.B, which shows its influence on the system in the
inteival u.u - 1.66 u.a. mL
-1
. The optimum value was close to u.1 u.a. mL
-1
.
Figure 4.C shows that the optimum inuocyanine gieen concentiation,
assayeu in the inteival u.u - 2u.8 µmol L
-1
, was S.2 µmol L
-1
. The effect of
the tempeiatuie on the system was stuuieu in the iange 1u - 4u °C,
obtaining only a slight ueciease on the analytical paiametei up to a
tempeiatuie of 22ºC, but it was shaipei at highei tempeiatuies. Thus, a
tempeiatuie of 2uºC was chosen.










Capítulo 3

186
Table 1. 0ptimization of vaiiables
Type of
vaiiable
vaiiable
Range
stuuieu
0ptimum
value
Instiumental
Excitation wavelength (nm) Suu - 8uu 764
Emission wavelength (nm) 78u - 8Su 8u6
Ex¡em slits (nm) 4 - 16 4¡4
Powei eneigy (v) 4uu-8uu 7uu
Aveiage time (s) u.u12 - u.1S u.1
Stoppeu-flow
uevice
Reaction volume (mL) u.1 - u.S u.S
Tempeiatuie (ºC) S - 4u 2u
Chemical
pB 2 - 9 7.S
|Tiisj

buffei (mmol L
-1
) 1u - 2uu 2S
|AuNPsj (µmol L
-1
)
u.u - S2.4 S9.4
|inuocyanine gieenj (µmol L
-1
)
u.u - 2u.8 S.2
|laccasej (u.a. mL
-1
) u.u - 1.66 u.1


2.S. Analytical featuies

Calibiation giaphs foi five polyphenols (CA, BY, BB, uA, PY) weie
iun unuei the optimal expeiimental conuitions summaiizeu in Table 1,
using the inuuction time above uefineu as the analytical paiametei, anu
assaying each stanuaiu in tiiplicate. Table 2 summaiizes the figuies of
meiit of the methou, incluuing the calibiation equations, lineai ianges,
L0Bs anu piecision uata. The L0Bs obtaineu, calculateu accoiuing to I0PAC
iecommenuations |2uj, iangeu between u.u1 anu u.u4 µmol L
-1
. These
values aie lowei than most of the L0Bs uesciibeu foi uA using
ampeiometiic biosensois, which iange between u.19 anu Su µmol L
-1

|7,1u,11,1Sj. The L0Bs weie also stuuieu in the absence of AuNPs, with the
aim of checking theii influence on the featuies of the methou, obtaining
values about thiee-times highei. The stuuy of the effect of ascoibic aciu (2.u
µmol L
-1
)

on the system showeu that the analytical paiametei was not
affecteu. The piecision, expiesseu as ielative stanuaiu ueviation (RSB %)
was stuuieu at two concentiations of each analyte, u.2 anu 1 µmol L
-1
. The
values (n = 11) iangeu between 1.8 anu S.6 %.

Innovaciones en la determinación no cromatográfica de antioxidantes en alimentos

187































Figure 4. Stuuy of the influence of AuNPs (A), laccase (B) anu inuocyanine
gieen (C) concentiations on the analytical paiametei in the
piesence of 2 µmol L
-1
uA. |AuNPsj = 4u.S µmol L
-1
on Figuies B
anu C; |laccasej = u.S u.a. mL
-1
on Figuie A anu u.1 u.a. mL
-1
on
Figuie C; |inuocyanine gieenj = 1u.4 µmol L
-1
on Figuie A anu B;
tempeiatuie = 2u ºC, pB = 7.S anu |Tiisj = 2S mmol L
-1
in all
instances.
0
50
100
150
200
250
0 10 20 30 40 50 60
T
i
m
e
,

s

[Au NPs], µmol L
-1


0
100
200
300
400
500
0 0,5 1 1,5 2
T
i
m
e
,

s

[Laccase], u.a. mL
-1

0
100
200
300
400
500
600
0 5 10 15 20 25
T
i
m
e
,

s

[Indocyanine Green], µmol L
-1

C
a
p
í
t
u
l
o

3



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.

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A
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(
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)

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/
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(
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(
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)

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%

(
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)

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+

(
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.
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±

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.
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±

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)

x

+

(
2
3

±

2

0
,
9
9
9
0
.
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1
7


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1

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e

c
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e
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t
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t
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n

(
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m
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l

L
-
1
)
.

2

R
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s
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.

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f

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o
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d

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)
(
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)

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o
n
c
.

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o
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d

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S
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)
(
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R
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c
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r
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%

(
2
)






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s
t

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r
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9
4

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0

3
4
4

±

1
1
6

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0
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0

1
0
0
.
2

2

S
a
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9
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4
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8

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5

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.
9

4

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s

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e
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a
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8

±

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4
8

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6

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.
4

9
7
.
3

(
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)

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o
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y
p
h
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A
,

c
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r
a
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-
1

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d
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i
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µ
m
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l

L
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v
e
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y
.


Innovaciones en la determinación no cromatográfica de antioxidantes en alimentos

189
2.4. Application of the methou

The pioposeu methou was applieu to the analysis of foui
commeicial fooustuffs, oiange juice, ieu giape juice anu teas, to ueteimine
theii coiiesponuing polyphenol content, which was expiesseu as uA
concentiation. The samples weie analyseu following the pioceuuie above
uesciibeu. Each sample was uiluteu with uistilleu watei to match the lineai
iange of the calibiation giaph anu analyseu using both the pioposeu anu
the Folin-Ciocalteu official methous. The methou was testeu in two ways: 1)
by ueteimining the polyphenolic concentiation in the samples, anu 2) by
stuuying the iecoveiy aftei auuition of two stanuaiu aliquots of uA at two
concentiation levels (u.2 anu 1µmol L
-1
) which coveieu the lineai iange of
the calibiation giaph. The iesults achieveu aie summaiizeu in Table 3,
which incluue the commeicial name of the samples, the fooustuff type, the
concentiations obtaineu by both iefeience anu pioposeu methous anu the
iecoveiy uata. As can be seen, the iesults obtaineu by the pioposeu methou
aie slightly lowei than those obtaineu by the iefeience methou, which
coulu be asciibeu to the lowei selectivity of the latei. The iecoveiy
peicentages, calculateu by subtiacting the iesults obtaineu fiom similai
unspikeu samples, iangeu between 89.9 anu 1uu.2 %.

3. Conclusions

The new appioach uesciibeu, involving kinetic methouology, long
wavelength fluoiescence measuiements anu AuNPs, has shown its
usefulness foi the ueteimination of polyphenol compounus in foou
samples. Stoppeu-flow mixing technique is useu foi the fast anu automatic
mixtuie of the ieactants, which simplify the uevelopment of the methou.
Although the consume of enzyme is highei than in the methous involving
its immobilization, the enzyme amount iequiieu foi each measuiement is
veiy low owing to the high sensitivity of the methou. It has been also shown
that the activity of the enzyme is uecieaseu by its inteiaction with AuNPs,
which allows the impiovement of the L0Bs of the methou. The values
obtaineu aie lowei than those obtaineu in most of the methous baseu on
ampeiometiic biosensois |7,1u,11,1Sj. Also, the use of long wavelength
fluoiimetiy avoius potential backgiounu signals fiom the sample matiix,
which can appeai at lowei wavelengths.
Capítulo 3

190
References

|1j Ignat, I.; volf, I.; Popa, v. I.; Foou Chem. 2u11, 126, 1821-18SS.
|2j Robbins, R. }.; }. Agiic. Foou Chem. 2uuS, S1, 2866-2887.
|Sj Sies, B.; Aich. Biochem. Biophys. 2u1u, Su1, 2-S.
|4j Kalili, K. N.; Be villieis, A.; }. Sep. Sci. 2u11, S4, 8S4-876.
|Sj }ác, P.; Polásek, N.; Pospisilová, N.; }. Phaim. Biomeu. Anal. 2uu6, 4u,
8uS-814.
|6j Singleton, V. L.; Rossi, J. A.; Am. J. Enol. Viticult. 1965, 16,
144-158.
|7j ElKaoutit, N.; Naianjo-Rouiiguez, I.; Temsamani, K. R.; Beinánuez-
Aitiga, N. P.; Belliuo-Nilla, B.; Biualgo-Biualgo ue Cisneios, }. L.;
Foou Chem. 2uu8, 11u, 1u19-1u24.
|8j A.B' Annibale, S.R. Stazi, v. vencigueiia, u.u. Seimanni, }. Biotech. 77
(2uuu) 26S-27S.
|9j uamella, N.; Campuzano, S.; Reviejo, A. }.; Pingaiion, }. N.; }. Agiic.
Foou Chem. 2uu6, S4, 796u-7967.
|1uj Nonteieali, N. R.; Bella Seta, L.; vastaiella, W.; Pilloton, R.; }. Nol.
Cat. B Enzym. 2u1u, 64, 189-194.
|11j ElKaoutit, N.; Naianjo-Rouiiguez, I.; Temsamani, K. R.; Bominguez
ue la vega, N.; Biualgo-Biualgo ue Cisneios, }. L.; }. Agiic. Foou
Chem. 2uu7, SS, 8u11-8u18.
|12j Shiauuha; Shekhei, R.; Sehgal, S.; Kamthania, N.; Kumai, A.; Enzyme
Res. 2u11, IB 217861, uoi: 1u.4u61¡2u11¡217861.
|1Sj Bi Fusco, N.; Toitolini, C.; Beiiu, B.; Nazzei, F.; Talanta, 2u1u, 81,
2SS-24u.
|14j Ibaiia-Escutia, P.; }uaiez-uomez, }.; Calas-Blanchaiu, C.; Naity, }. L.;
Ramiiez-Silva, N. T.; Talanta, 2u1u, 81, 16S6-1642.
|1Sj Chawla, S.; Rawal, R.; Shabnam; Kuhau, R. C.; Punuii, C. S.; Anal.
Nethous, 2u11, S, 7u9-714.
|16j Zhang, L.; Yuan, R.; Chai, Y.; Li, X.; Anal. Chim. Acta, 2uu7, S96, 99-
1uS.
|17j Tuikevich, }.; Stevenson, P. C.; Billiei, }.; Biscuss. Faiauay Soc. 19S1,
11, SS-7S.
|18j Bagys, N.; Babeiska, K.; Shleev, S.; Ainebiant, T.; Kulys, }.; Ruzgas,
T.; Electiochem. Commun. 2u1u, 12, 9SS-9SS.
|19j Piontek, K.; Antoiini, N.; Choinowski, T.; }. Biol. Chem. 2uu2, 277,
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Innovaciones en la determinación no cromatográfica de antioxidantes en alimentos

191
|2uj Long, u. L.; Winefoiunei, u. B.; Anal. Chem. 198S, SS, 712A-724A.













CAPITULO 4
NUEVAS INVESTIGACIONES CON
NANOPARTICULAS DE ORO


















Nuevas Investigaciones con nanopaiticulas ue oio

19S
1. Introducción

Con objeto ue auquiiii nuevos conocimientos sobie la sintesis y
caiacteiizacion ue las nanopaiticulas ue oio (AuNPs) y sobie el uso ue
técnicas instiumentales en las que se utilizan estas NPs, se iealizo una
estancia bieve ue ties meses en el "Institute of Analytical Chemistiy,
Chemo- anu Biosensois" ue la 0niveisiuau ue Regensbuig (Bavieia,
Alemania), bajo la tutela uel Piofesoi 0tto Wolfbeis.

La iealizacion ue esta estancia ha peimitiuo también cumplii uno
ue los iequisitos necesaiios paia pouei optai a la Nencion ue Boctoiauo
Euiopeo.


2. Objetivos

Se planteaion los uos objetivos siguientes:

1) Sintesis ue AuNPs a paitii ue antioxiuantes ue uso alimentaiio.
2) Besaiiollo ue un sensoi selectivo ue AuNPs basauo en la iesonancia
uel plasmon ue supeificie (SPR).


3. Conocimientos adquiridos

S.1. Sintesis ue nanopaiticulas ue oio

Entie touos los métouos convencionales ue sintesis ue AuNPs
basauos en la ieuuccion ue ueiivauos ue oio(III), el más populai ue ellos ha
siuo él que emplea como agente ieuuctoi citiato souico sobie BAuCl
4
en
meuio acuoso, intiouuciuo poi Tuikevich en 19S1 |1j. Es un métouo
sencillo que iequieie solamente un equipo ue ieflujo uonue se calienta y
mantiene a una tempeiatuia constante una uisolucion ue BAuCl
4
, a la que
se auiciona una cantiuau auecuaua ue una uisolucion ue citiato souico. La
uisolucion inicialmente amaiilla va viianuo a azul-violeta y finalmente,
uespués ue unos Su minutos, piesenta coloiacion ioja intensa uebiuo a la
piesencia ue AuNPs ue unos 2u nm ue uiámetio. Posteiioimente en 197S
Fiens intento obtenei nanopaiticulas ue un tamaño pieueteiminauo (entie
Capitulo 4

196
16 y 147 nm) contiolanuo su foimacion. Paia ello piopuso un métouo
uonue la ielacion citiato tiisouico - Au(III) iba vaiianuo según el uiámetio
ueseauo. En 1999 Kunitake uesaiiollo un métouo paia obtenei AuNPs
estabilizauas poi S-meicaptopiopionato ue souio, en uonue se piouuce la
auicion simultanea ue la sal ue citiato y ue un suifactante anfifilico (Figura
1); el tamaño ue la nanopaiticula pueue sei contiolauo meuiante la
vaiiacion ue la ielacion estabilizauoi¡oio |2j.


Fig 1. Sintesis ue Kunitake.


En la investigacion iealizaua se utilizo la sintesis ue Tuikevitch ya
que es muy sencilla y iepiouucible. Las nanopaiticulas fueion
caiacteiizauas meuiante fotometiia y micioscopia TEN.


S.2. Técnica ue iesonancia uel plasmon ue supeificie (SPR)

Besue su piimeia obseivacion a piincipios uel siglo XX, el fenomeno
fisico ue la iesonancia uel plasmon ue supeificie (SPR) ha encontiauo un
lugai uestacable en aplicaciones ue tipo piáctico tales como el uesaiiollo ue
uetectoies sensibles, que iesponuen a la ausoicion ue maciomoléculas
sobie su supeificie. Posteiioimente se obseivo un pation "anomalo" ue
banuas oscuias y claias en la luz ieflejaua, cuanuo se hizo inciuii un haz ue
luz polaiizaua en un espejo con una ieu ue uifiaccion en su supeificie. La
inteipietacion fisica uel fenomeno fue iniciaua poi Rayleigh, peio no fue
hasta 1968, cuanuo 0tto, Kietschmann y Raethei uescubiieion la
excitacion ue los plasmones ue supeificie |S,4j. La aplicacion ue sensoies
Nuevas Investigaciones con nanopaiticulas ue oio

197
basauos en SPR paia monitoiizai inteiacciones biomoleculaies fue
uemostiaua poi piimeia vez en 198S poi Lieubeig |Sj.

Paia explicai la excitacion ue los plasmones ue supeificie, pueue
utilizaise el montaje expeiimental ue la Figura 2. 0n haz ue luz polaiizaua
se hace inciuii a tiavés ue un piisma sobie un chip sensoi iecubieito con
una uelgaua pelicula ue oio, la cual actúa como un espejo ieflejanuo la
iauiacion. Al ii cambianuo el ángulo ue inciuencia y monitoiizai la
intensiuau ue la luz ieflejaua, ésta alcanza un valoi minimo (Figura 2, línea
A), cuanuo la iauiacion inciuente excita el plasmon ue supeificie
inuucienuo su iesonancia. Los fotones ue la luz p-polaiizaua pueuen
inteiactuai con los electiones libies ue la pelicula metálica, piouucienuo
oscilaciones en foima ue ola ue los electiones libies y ieuucienuo ue este
mouo la intensiuau ue la luz ieflejaua.

El ángulo al cual ocuiie la máxima péiuiua ue la intensiuau ue la
iauiacion ieflejaua se conoce como ángulo ue iesonancia o ángulo SPR. Este
ángulo uepenue ue las caiacteiisticas opticas uel sistema, tales como los
inuices ue iefiaccion ue los meuios situauos a ambos lauos ue la pelicula ue
oio. Nientias se mantiene constante el inuice ue iefiaccion en el lauo uel
piisma, se piouuciiá un cambio en el inuice ue iefiaccion en la supeificie
uel metal cuanuo se ueposite algún tipo ue masa sobie ella (poi ejemplo
pioteinas). Poi lo tanto, las conuiciones a las que se piouuce la iesonancia
uel plasmon ue supeificie pueuen cambiai, piouuciénuose un
uesplazamiento uel ángulo SPR (Figura 2, línea B), que pueue utilizaise
paia obtenei infoimacion ue la cinética ue un pioceso, poi ejemplo la
ausoicion ue pioteinas en la supeificie uel metal.

La técnica SPR es una opcion excelente paia monitoiizai los
cambios uel inuice ue iefiaccion en las inmeuiaciones ue la supeificie uel
metal. La Figura 3 muestia la vaiiacion uel ángulo al cual se obtiene el
minimo con el tiempo, iepiesentacion a la que se le uenomina
sensoigiama. Cuanuo el inuice ue iefiaccion cambia, el ángulo al cual se
obtiene el minimo ue intensiuau se uesplazaiá, como pueue obseivaise en
uicha figuia, uonue (A) muestia la iepiesentacion ieal ue la intensiuau ue
la luz ieflejaua fiente al ángulo inciuente y (B) es la iepiesentacion uespués
uel cambio en el inuice ue iefiaccion. El pioceso ue ausoicion-uesoicion
pueue sei monitoiizauo en tiempo ieal y las cantiuaues ue las especies
Capitulo 4

198
ausoibiuas pueuen sei ueteiminauas. La técnica SPR no solo es auecuaua
paia meuii la uifeiencia entie estos uos estauos, si no que también se
pueue monitoiizai el cambio en el tiempo, si se monitoiiza el tiempo que
uuia el uesplazamiento uel ángulo ue iesonancia al cual el minimo es
obseivauo.



Figura 2. Nontaje expeiimental SPR. 0n chip sensoi con un
iecubiimiento ue oio es colocauo en un piisma. La fuente ue luz
(estiella) emite una iauiacion polaiizaua e inciue sobie el chip sensoi.
La intensiuau ue la luz ieflejaua es meuiua en el uetectoi (uisco). A un
ueteiminauo valoi uel ángulo ue inciuencia (θ) , ocuiie la excitacion uel
plasmon ue supeificie, lo que conlleva una uisminucion en la intensiuau
ue la luz ieflejaua (A). 0n cambio en el inuice ue iefiaccion en la
supeificie ue la pelicula ue oio causaiá un uesplazamiento en el ángulo
ue A a B.


Si el cambio obseivauo es uebiuo a una inteiaccion moleculai, la
cinética ue esta inteiaccion pueue sei meuiua en tiempo ieal. Los sensoies
SPR miuen solamente una iegion o volumen muy limitauo en la supeificie
uel metal. La piofunuiuau ue la penetiacion uel campo electiomagnético
(también llamauo campo evanescente) en la pelicula ue oio a la cual se
obseiva la señal, noimalmente no exceue unos pocos cientos ue
nanometios.





(θ)
(θ)
Nuevas Investigaciones con nanopaiticulas ue oio

199




Figura 3. Esquema ue un sensoigiama en el que se monitoiiza el ángulo al cual el
minimo es obseivauo fiente al tiempo. Al piincipio no ocuiie ningún
cambio en el sensoi obtenienuo una linea ue base paia el minimo en el
ángulo SPR (A). Bespués ue la inyeccion ue una muestia (flecha) las
biomoleculas se ausoibeián en la supeificie iesultanuo un cambio en el
inuice ue iefiaccion y un uesplazamiento en el ángulo SPR hacia la
posicion B.

0na limitacion ue los sensoies SPR es su falta ue selectiviuau
intiinseca ya que touos los cambios en el inuice ue iefiaccion en el campo
evanescente seián ieflejauos en un cambio en la señal. Estos cambios en el
inuice ue iefiaccion pueuen uebeise, poi ejemplo, a vaiiaciones en la
composicion o concentiacion ue la uisolucion iegulauoia. También, la
ausoicion ue mateiial sobie la supeificie uel sensoi pueue afectai al inuice
ue iefiaccion. La cantiuau ue especies ausoibiuas pueue sei ueteiminaua
uespués ue la inyeccion ue la uisolucion iegulauoia inicial que oiigino la
linea ue base, como se muestia en la Figura 3. Paia conseguii la ueteccion
selectiva en un sensoi ue SPR, su supeificie uebe sei mouificaua con
liganuos auecuauos que ieaccionen o inteiaccionen selectivamente con los
compuestos ue inteiés, peio que no faciliten la ausoicion ue otios
componentes piesentes en la muestia o ue la uisolucion iegulauoia.

En el caso más simple ue una meuiua ue SPR, el analito es captuiauo
poi el liganuo, poi ejemplo un anticueipo (Figura 4), el cual está
inmovilizauo en la supeificie uel sensoi pieviamente a la meuiua. Existen
numeiosos sensoies con uifeientes liganuos inmovilizauos que son
comeicialmente uisponibles, aunque también se pueue inmovilizai el
liganuo fácilmente en el laboiatoiio. En el caso más sencillo, uenominauo
Capitulo 4

2uu
ueteccion uiiecta, la captuia ue la sustancia analizaua poi el liganuo ua
lugai a una señal meuible. La Figura 5 muestia la señal uel sensoi paso a
paso en el ciclo ue meuicion con ueteccion uiiecta.

















Figura 4. Repiesentacion esquemática ue la ueteccion uiiecta: el analito es
captuiauo poi los liganuos (Y) inmovilizauos en la supeificie uel
sensoi, poi ejemplo, un hiuiogel. La acumulacion ue los analitos ua
lugai a un cambio uel inuice ue iefiaccion en el campo evanescente
cambianuo el ángulo ue SPR.

Caua meuiua se inicia con el aconuicionamiento ue la supeificie uel
sensoi utilizanuo una uisolucion iegulauoia auecuaua (1). 0n aspecto
ciitico es la uisponibiliuau ue una iefeiencia fiable antes ue que se inicie la
meuiua. En este punto, la supeificie uel sensoi contiene los liganuos
activos, piepaiauos paia captuiai los analitos. Al inyectai la solucion que
contiene los analitos (2), éstos son captuiauos en la supeificie. Aunque
otios componentes ue la muestia pouiian auheiiise a la supeificie uel
sensoi, pouiian eliminaise fácilmente ya que no existiiia un enlace
selectivo. En este paso, la cinética ue ausoicion uel analito pueue sei
ueteiminaua en tiempo ieal. A continuacion, se inyecta uisolucion
iegulauoia en el sensoi y los componentes que no están enlazauos
selectivamente son uesplazauos (S). Como se inuica en la Figura 5, la
cantiuau acumulaua ue analito se pueue ueteiminai meuiante la iespuesta
Nuevas Investigaciones con nanopaiticulas ue oio

2u1
uel SPR (AR). También se pueue piouucii en este paso la uisociacion ue la
sustancia analizaua, lo que peimite estuuiai la cinética uel pioceso ue
uisociacion. Finalmente, se inyecta una uisolucion ue iegeneiacion, la cual
iompe la union selectiva entie el analito y el liganuo (4). Si se ha hecho una
auecuaua inmovilizacion ue los liganuos, éstos peimaneceián en el sensoi,
mientias que los analitos son cuantitativamente eliminauos. Esta etapa es
básica paia iealizai múltiples meuiuas con el mismo chip sensoi ya que la
utilizacion ue una uisolucion iegeneiante que mantenga a los ligánuos
activos peimitiiá iealizai cientos e incluso miles ue meuiuas. La etapa ue
iegeneiacion meuiante la uisolucion iegeneiauoia peimite aconuicionai la
supeificie uel sensoi paia el siguiente análisis. Si la iegeneiacion es
incompleta, la masa acumulaua piovocaiá un aumento en el nivel ue
iefeiencia.

Figura 5. Sensoigiama que muestia las etapas ue un ciclo ue análisis: 1, la
uisolucion aconuicionauoia está en contacto con el sensoi (linea ue
base); 2, inyeccion ue la uisolucion ue la muestia (asociacion); S,
inyeccion ue la uisolucion iegulauoia (uisociacion); AR inuica la
iespuesta meuiua uebiuo al analito; 4, inyeccion ue la uisolucion
iegeneiante paia la eliminacion ue los analitos ieteniuos (iegeneiacion),
y piepaiacion uel sistema paia otio ciclo ue meuiua. Como pueue
obseivaise, en t
1
se piouuce un cambio en el inuice ue iefiaccion.
D
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t
o

d
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S
P
R
Capitulo 4

2u2
Fiecuentemente, las meuiciones ue SPR se utilizan paia estuuiai la
cinética ue un pioceso ue enlace, poi lo que es básico paia obtenei
iesultauos ieales que el pioceso ue inmovilizacion uel liganuo no afecte a
su afiniuau hacia el analito.

Auemás ue la iealizacion ue estuuios cinéticos y teimouinámicos,
las meuiuas SPR también pueuen sei utilizauas paia la ueteiminacion ue la
concentiacion ue un analito en una muestia (análisis cuantitativo). En este
caso, en piimei lugai se uebe obtenei la calibiacion miuienuo las señales
paia uistintas concentiaciones ue uisoluciones estánuai uel analito. Los
iesultauos obteniuos al supeiponei los uifeientes sensoigiamas se
muestian en la Figura 6, en la que se obseiva que las mesetas uebiuas a la
etapa ue asociacion van aumentanuo al aumentai la concentiacion uel
analito |6j. Se pueue constiuii una iecta ue calibiauo iepiesentanuo la
iespuesta analitica (AR) uespués ue un cieito inteivalo ue tiempo (t
1
)
fiente a la concentiacion.

Paia ueteiminai la concentiacion uesconociua ue un analito en una
muestia pueuen iealizaise uiluciones secuenciales, poi ejemplo poi 1u,
1uu y 1uuu. Paia ueteiminaciones más piecisas pueuen utilizaise
uiluciones que vaiien en un factoi ue 2. Si la concentiacion uel analito en la
muestia es muy alta, la muestia sin uiluii uaiá iesultauos poi encima ue la
zona supeiioi ue la iecta ue calibiauo. Las uisoluciones uiluiuas, sin
embaigo, uaián lugai a señales situauas en la zona infeiioi ue la iecta ue
calibiauo y se pouiá ueteiminai la concentiacion uel analito.

Como se menciono anteiioimente, la técnica SPR uetecta los
cambios que se piouucen en el inuice ue iefiaccion en la supeificie uel
sensoi uebiuos a la cantiuau ue masa uepositaua. La ueteccion uiiecta solo
es posible si la ietencion uel analito piouuce cambios meuibles uel inuice
ue iefiaccion. Esta situacion se consigue con mayoi faciliuau si el peso
moleculai ue la sustancia ueteiminaua es elevauo (alieueuoi ue 1uuu Ba o
supeiioi). Sin embaigo, paia moléculas pequeñas, seiia necesaiio una
cantiuau elevaua ue las mismas paia piouucii un cambio meuible uel inuice
ue iefiaccion, poi lo que el análisis seiia poco sensible. Si el analito es una
molécula pequeña (peso moleculai<1uuu Ba), la ueteccion se iealiza
meuiante un foimato ue ensayo competitivo o ue inhibicion ue tipo
sánuwich. En touos los foimatos ue ensayo, no solo la minima
Nuevas Investigaciones con nanopaiticulas ue oio

2uS
concentiacion uetectable es limitaua, sino también el númeio fisico ue los
elementos que pueuen sei inmovilizauos en la supeificie uel sensoi, lo que
piopoiciona un valoi limite máximo.


Figura 6. Sensoigiamas obteniuos paia uistintas concentiaciones ue analito. }usto
uespués ue la inyeccion en t
u
se piouuce un enlace selectivo ue la
sustancia analizaua. El tianspoite ue masa a la supeificie es limitante ue
la velociuau y uepenue linealmente ue la concentiacion. La meuiua ue las
penuientes (uR ¡ ut) peimite constiuii la iecta ue calibiauo y, poi tanto,
pueue ueteiminai la concentiacion ue analito en una muestia. La fase ue
uisociacion comienza en t
1
, al inyectai la uisolucion uisociante,
piovocanuo la iuptuia uel enlace analito-liganuo. Los uatos obteniuos en
este pioceso pueuen utilizaise paia ueteiminai la constante ue
uisociacion (k
u
).


4. Trabajo experimental

La piimeia etapa uel tiabajo expeiimental iealizauo consistio en
apienuei a sintetizai AuNPs ue tamaño contiolauo utilizanuo
pioceuimientos bien estableciuos, asi como a utilizai la técnica SPR,
aspectos en los que el giupo ue investigacion uel Piofesoi Wolfbeis tiene
una gian expeiiencia. Posteiioimente se pioceuio a uesaiiollai los uos
objetivos planteauos que consistieion en:
1. Línea de
base
2. Asociación 3. Disociación 4. Regeneración 1. Línea de
base
Paso:
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Capitulo 4

2u4

1) Investigai la posibiliuau ue conseguii la sintesis ue AuNPs meuiante
pioceuimientos alteinativos a los ya estableciuos.
2) 0tilizai la técnica SPR paia la cuantificacion ue AuNPs.

Aunque el bieve tiempo uisponible no peimitio completai
satisfactoiiamente estos objetivos, cabe uestacai que el tiabajo
expeiimental iealizauo ha contiibuiuo positivamente a la foimacion
cientifica uel uoctoianuo.

A continuacion se uesciiben bievemente las investigaciones
iealizauas.


4.1. Sintesis ue nanopaiticulas ue oio a paitii ue antioxiuantes ue uso
alimentaiio.

Paitienuo ue la sintesis ue Tuikevich |1j, uonue se emplea citiato
souico como ieuuctoi uel BAuCl
4
paia la foimacion ue nanopaiticulas ue
oio, se planteo la posibiliuau ue utilizai otio tipo ue ieuuctoies paia llevai
a cabo esta sintesis. Como muestia la Figura 7, la utilizacion ue este
ieuuctoi peimite obtenei AuNPs con máximo ue absoicion a unos S2u nm
(Figura 7.1) y con un tamaño meuio ue paiticula pioximo a 2u nm (Figura
7.2). Como ieuuctoies alteinativos se ensayaion antioxiuantes
alimentaiios, tales como las sales souicas ue los galatos ue piopilo (E-S1u),
octilo (E-S11) y uouecilo (E-S12), butilhiuioxianisol (BBA) y
butilhiuioxitolueno (BBT).

Se iealizaion uifeientes ensayos con touos estos compuestos paia
pouei obtenei uisoluciones monouipeisas ue nanopaiticulas ue oio ue
tamaño contiolauo.

Se utilizaion cantiuaues vaiiables ue BAuCl
4
y antioxiuantes,
mouificanuo también la tempeiatuia y el tiempo ue sintesis. En ninguno ue
los ensayos iealizauos se consiguieion iesultauos satisfactoiios. No
obstante, en algunos casos se obseivo la foimacion ue nanopaiticulas ue
tamaño ielativamente gianue, asi como su agiegacion, ya que los espectios
ue absoicion obteniuos piesentaban banuas situauas a longituues ue onua
Nuevas Investigaciones con nanopaiticulas ue oio

2uS
mayoies que la banua caiacteiistica obteniua paia una uisolucion con
nanopaiticulas uispeisauas. Asimismo, las uisoluciones piesentaban una
coloiacion violeta-azulaua, inuicativo ue su elevauo tamaño.

Figura 7. Espectio ue absoicion (1) e imagen TEN obteniua poi un micioscopio
electionico Leo 912 AB (2) ue AuNPs meuiante una sintesis ue Tuikevich
empleanuo citiato souico como agente ieuuctoi.


4.2. Sensoi SPR

Con objeto ue uesaiiollai un sensoi SPR sensible a AuNPs, se
investigo la posibiliuau ue que la albúmina ue sueio bovino (BSA) foimaia
un enlace selectivo con las AuNPs. La piimeia etapa ue este estuuio fue la
inmovilizacion ue BSA en la supeificie ue la lámina ue oio uel sensoi. Paia
ello se ueposito una fina capa ue áciuo 16-meicaptohexauecanoico
(SB(CB
2
)
1S
C00B), ue foima que los giupos caiboxilicos inteiaccionasen
con la BSA. 0na vez anclauos los giupos caiboxilicos a la lámina ue oio, se
paso una coiiiente ue 1-etil-S-(uimetilaminopiopilcaibouiimiua) (EBC),
ieactivo que se usa geneialmente como un agente activauoi ue giupos
caiboxilicos paia el acople con aminas piimaiias que conuuzcan a enlaces
amiua. Al pasai una coiiiente ue BSA se piouujo su inmovilizacion
queuanuo piepaiauo el sistema paia estuuiai su iespuesta fiente a las
AuNPs.

Las AuNPs como eia ue pievei, se enlazaion a BSA, uanuo mayoies
señales al aumentai su concentiacion, obtenienuo asi la coiiesponuiente
iecta ue calibiauo. Sin embaigo, el enlace BSA-AuNPs eia uemasiauo fueite,
®CentralEM-LAB,UniversityHospitalRegensburg(2010) 300nm
(2)
Tamaño = 20 nm

Abs
= 529 nm
A
b
s
o
r
b
a
n
c
i
a
(nm)
(1)
Capitulo 4

2u6
ya que la señal uel SPR apenas uisminuia al pasai coiiientes ue lavauo con
agua o NaCl. La iegeneiacion se intento también hacienuo pasai uifeientes
coiiientes ue uisoluciones iegulauoias ue pBs entie 1,S y 1u,1, peio sin
iesultauo satisfactoiio.

El siguiente paso ue la investigacion fue el estuuio uel
compoitamiento uel sistema en ausencia ue BSA. Aunque las señales
obteniuas fueion menoies, también vaiiaion con la concentiacion ue
AuNPs uanuo lugai a una iecta ue calibiauo con un inteivalo uinámico y
coeficiente ue iegiesion similai a los obteniuos en piesencia ue BSA,
aunque la iepiouucibiliuau ue las meuiuas fue peoi.

Este hecho pone ue manifiesto la existencia ue enlaces inespecificos
cuanuo se utilizala BSA. Se ensayo una alteinativa más selectiva paia
enlazai las AuNPs, usanuo cisteina, un u-aminoáciuo con giupo tiol afin al
oio. El estuuio se inicio iecubiienuo la supeificie ue la lámina ue oio uel
SPR con una capa ue BS-(CB
2
)
1S
-C00B y BS-(CB
2
)
1S
-CB
S
, en uifeientes
piopoiciones paia uetectai la posible existencia ue enlaces inespecificos.
0na vez seleccionaua la más auecuaua, se activaion los giupos caiboxilo
con EBC paia su union con la N-hiuioxisulfosuccinimiua (Sulfo-NBS) y poi
último se enlazo la cisteina que seiia la encaigaua ue ietenei a las AuNps.
Sin embaigo no se obtuvo ningún iesultauo satisfactoiio ya que no se puuo
inmovilizai la cisteina auecuauamente.

Se ensayo una estiategia alteinativa a la utilizaua en la expeiiencia
anteiioi, en la que se hacia fluii una coiiiente ue cisteina sobie la lámina ue
oio uel sensoi uuiante unos minutos. Consiueianuo que piobablemente la
cinética ue la ieaccion fueia lenta y no se piouujeia el enlace uebiuo al
cauual ue la coiiiente, se utilizo un compuesto con uoble giupo tiol (BS-
(C
1S
)-C00NBS), ue tal maneia que poi un extiemo se unieia a la lámina ue
oio y el otio extiemo tiol queuase libie paia ieaccionai con la cisteina. En
esta ocasion en vez ue pasai una coiiiente ue cisteina, la lámina se
sumeigio en la uisolucion uuiante S h, paia favoiecei asi la fijacion ue la
cisteina sobie la supeificie uel sensoi (Figura 8).




Nuevas Investigaciones con nanopaiticulas ue oio

2u7


Figura 8. Esquema ue la estiategia seguiua paia la inmovilizacion ue Au-NPs


Las bajas señales SPR obteniuas pusieion ue manifiesto que las
AuNps no se inmovilizaban en la cisteina o que la inteiaccion no se
piouucia poique la cinética fueia lenta. Incluso pasanuo la uisolucion ue
nanopaiticulas a cauuales bajos no se mejoio la señal. Como alteinativa, en
lugai ue pasai una coiiiente ue nanopaiticulas, se uejo un pequeño
volumen ue uisolucion en contacto con uno ue los canales uel sensoi
uuiante 48 hoias, mientias que el otio canal se uejo como iefeiencia. Si
existieia ueposito ue nanopaiticulas se uetectaiia compaianuo los minimos
ue iesonancia ue ambos canales. Lamentablemente las meuiuas uieion
valoies casi iuénticos, lo que inuico que no existe inmovilizacion.

La falta ue iespuesta uel sistema ensayauo pouiia atiibuiise a que,
al inmovilizai el compuesto con uoble giupo tiol, ambos tioles teiminales
se enlazaian a la lámina ue oio, impiuienuo su inteiaccion con las AuNPs.












Capitulo 4

2u8
Bibliografía

|1j Tuikevitch, }.; Stevenson, P. C.; Billiei, }.; Biscuss. Faiauay Soc.
19S1, 11, SS-7S.
|2j Yonezawa, T.; Kunitake, T.; Colloius Suif. A: Physicochem. Eng.
Asp. 1999, 149, 19S-199.
|Sj A. 0tto, Z. Phys., 1968, 216, S98-41u.
|4j E. Kietschmannanu, B. Reathei, Z. Natuifoisch. Teil A, 1968, 2S,
21SS-21S6.
|Sj B. Lieubeig, C. Nylanuei anu I. Lunustiom, Sens. Actuatois, 198S,
4, 299-Su4.
|6j S. ueib, u. Sanuoz, K. Nabiouk, A. Natavel, P. Naichot, T. Boshi, N.
villaz, N. Ronjat, R. Niquelis, C. Leveque anu N. ue Waaiu,;
Biochem. }., 2uu2, S64, 28S-292.











CAPITULO 5
DISCUSIÓN DE LOS RESULTADOS
DISCUSSION OF THE RESULTS






































Biscusion ue los iesultauos
211
Introducción

En este capitulo se aboiua una uiscusion uetallaua ue los iesultauos
obteniuos a lo laigo ue esta Tesis Boctoial. Se ponuián ue manifiesto los
puntos fueites y uébiles ue las metouologias uesaiiollauas asi como la
compaiacion con otias metouologias empleauas en el ámbito cientifico
paia la misma finaliuau.

Este capitulo se uiviue en uos secciones, uiscutienuo en la piimeia
los iesultauos obteniuos con las metouologias uesaiiollauas basauas en la
sepaiacion ciomatogiáfica, ueiivatizacion y ueteccion fluoiescente, y en
la segunua las metouologias automáticas no ciomatiogiáficas piopuestas
meuiante el uso ue la técnica ue flujo ueteniuo y nanopaiticulas.

1. Metodologías basadas en separación cromatográfica,
derivatización postcolumna y detección fluorescente

En este bloque se encuauian los uifeientes métouos
ciomatogiáficos uesaiiollauos paia la ueteiminacion ue compuestos
fenolicos en muestias ue oiigen alimentaiio. El objetivo geneial ue estos
métouos fue el establecimiento ue nuevas metouologias que peimitieian
mejoiai las piopieuaues analiticas básicas (piecision, selectiviuau y
sensibiliuau) y complementaiias (iapiuez, costes y factoies peisonales)
ue los métouos ya existentes, ue foima que los nuevos métouos
piopuestos pueuan sei una alteinativa ieal paia la ueteiminacion ue
estos compuestos ue gian inteiés en el contiol ue la caliuau alimentaiia.

Paia alcanzai el objetivo piopuesto se ha utilizauo la ciomatogiafia
ue liquiuos como técnica ue sepaiacion y la luminiscencia como sistema
ue ueteccion. No obstante, la innovacion ue los métouos piopuestos
iauica en los uiveisos sistemas ue ueiivatizacion post-columna
empleauos en caua uno ue ellos paia pouei uotai a los analitos (caientes
ue fluoiescencia nativa) ue piopieuaues luminiscentes. Asi, se han
uesaiiollauo:

• 0n métouo ue análisis que emplea un sistema ue ueiivatizacion
post-columna basauo en la foimacion ue quelatos luminiscentes
Capitulo S
212
ue los analitos con teibio(III) en meuio ligeiamente básico y en
piesencia ue T0P0 y AEBT.

• 0n métouo ue análisis que hace uso ue un sistema ue
ueiivatizacion basauo en la capaciuau ue los analitos paia
foimai complejos estables con aluminio(III) y teibio(III) en un
meuio submicelai oiiginauo poi SBS.

• 0n métouo ue análisis basauo en un sistema ue ueiivatizacion
uonue los analitos inteiaccionan con ceiio(Iv) y un fluoiofoio
ue laiga longituu ue onua.


Touas las metouologias uesaiiollauas en esta seccion compaiten
una seiie ue elementos comunes que apoitan uiveisas ventajas fiente a
otios métouos ue análisis ya estableciuos.

En piimei lugai, la columna empleaua paia la sepaiacion en los ties
métouos piopuestos es una columna monolitica C
18
, como alteinativa a las
columnas ue fase ieveisa empaquetauas con miciopaiticulas ue silice más
fiecuentemente utilizauas en la sepaiacion ue compuestos fenolicos. El
uso ue este tipo ue columnas peimite tiabajai con valoies ue cauual ue
fase movil más altos sin que ello iepeicuta negativamente en la piesion
ejeiciua en el sistema. Poi tanto, ofiece la posibiliuau ue tiabajai con
volúmenes ue inyeccion ue muestia ielativamente elevauos, obtenienuo
una mejoi señal analitica ya que no se piouuce el ensanchamiento ue los
picos ciomatogiáficos sino que se obtienen picos más estiechos y altos.
0tia ventaja que apoita este tipo ue columnas es la posibiliuau ue la
inyeccion uiiecta ue la muestias o, en touo caso, la utilizacion ue una baja
uilucion ya que la iegeneiacion ue la columna pueue haceise a una
elevaua velociuau.

En segunuo lugai, el uetectoi empleauo en touos los casos es un
espectiofluoiimetio paia obtenei señales analiticas a laiga, o
ielativamente laiga, longituu ue onua utilizanuo un fluoiofoio auecuauo o
la foimacion ue quelatos luminiscentes ue los analitos con teibio(III),
iespectivamente. La ueteccion en esta zona uel espectio peimite
minimizai posibles inteifeiencias uebiuas a la matiiz ue la muestia, ya
Biscusion ue los iesultauos
21S
que suelen emitii a menoies longituues ue onua. Auemás, la selectiviuau
que ofiece la luminiscencia sensibilizaua facilita la iuentificacion ue
algunos compuestos que no pueuen sei iuentificauos fácilmente usanuo
ueteccion fotométiica.

A continuacion se uiscutiiá en mayoi piofunuiuau los iesultauos
obteniuos paia caua una ue las metouologias uesaiiollauas.

Beteiminacion ue compuestos fenolicos en vino meuiante luminiscencia
sensibilizaua ue teibio.

Este métouo peimite la ueteiminacion simultánea, empleanuo
uoble ueteccion fotométiica y fluoiimétiica, ue 18 compuestos fenolicos
uifeientes entie los que se encuentian áciuos hiuioxibenzoicos e
hiuioxicinámicos, catequinas, aluehiuos fenolicos, flavonoiues y
estilbenos. El métouo se basa en la sepaiacion ciomatogiáfica y
ueiivatizacion post-columna ue los analitos paia tiansfoimai a uiez ue
ellos en ueiivauos fluoiescentes. La ueiivatizacion utiliza foimacion ue
quelatos solubles y estables ue los analitos-liganuos con teibio(III), en los
que el liganuo se excita absoibienuo la iauiacion y, a continuacion,
tiansfieie la eneigia absoibiua al lantániuo, el cual finalmente la emite en
foima ue eneigia luminosa.

Las ventajas uel métouo piesentauo fiente a otios métouos
pieviamente uesciitos son las siguientes:

• Las ueiivauas uel uso ue una columna monolitica C
18
, que se han
comentauo con anteiioiiuau.
• La ielativa laiga longituu ue onua a la que emite el teibio(III) pueue
minimizai posibles inteifeiencias existentes en la matiiz ue la
muestia, que suelen piouuciise a menoies longituues ue onua.
Auemás, la selectiviuau ue la luminiscencia sensibilizaua pueue
facilitai la iuentificacion ue algunos compuestos que no pueuen
uetectaise fácilmente meuiante fotometiia.
• El tiatamiento ue muestia es piácticamente nulo, iequiiienuo solo
la uilucion ue la muestia.
• Se ha uesciito poi piimeia vez el uso ue teibio(III) como ieactivo
paia la ueteiminacion ue aluehiuos aiomáticos.
Capitulo S
214
Estas caiacteiisticas han posibilitauo la sepaiacion ciomatogiáfica
en menoi tiempo que el iequeiiuo en otios métouos uesciitos paia estos
compuestos fenolicos, sepaianuo 1S compuestos fenolicos en muestias ue
vino en tan solo 2S minutos. Poi el contiaiio, otios métouos |1-2j que
emplean columnas empaquetauas logian la sepaiacion ue 14 a 2S fenoles
en 6u minutos, o S2 compuestos en 9u minutos. También ha peimitiuo
mejoiai los limites ue ueteccion, como se muestia en la Tabla 1, uonue se
compaian los limites ue ueteccion obteniuos fiente a los uesciitos en otios
métouos pieviamente publicauos |2-Sj. Se pueue obseivai que en el 4u%
ue los valoies compaiauos el limite ue ueteccion fluoiimétiico es menoi
que el fotométiico, poi lo que no solamente la luminiscencia sensibilizaua
ayuua a aumentai la selectiviuau si no también la sensibiliuau uel métouo.
Al compaiai el métouo piopuesto con otios métouos se pueue concluii que
se mejoian los niveles ue ueteccion fotométiicos y fluoiimétiicos en la
mayoiia ue los casos, ponienuo ue manifiesto la utiliuau uel métouo
uesaiiollauo.


Tabla 1. Compaiacion ue los L0Bs entie uifeientes métouos

Nétouo piopuesto |2j |Sj
Fotometiia Fluoiimetiia Fotometiia Fotometiia Fluoiimetiia
Analito L0B
(ng mL
-1
)
L0B
(ng mL
-1
)
L0B
(ng mL
-1
)
L0B
(ng mL
-1
)
L0B
(ng mL
-1
)
Aciuo gálico S S 14 6u -
Aciuo
piotocateuico
S6 2 18 12u -
Aciuo
p-hiuioxibenzoico
22 1S - - -
Aciuo salicilico 14 14 - - -
Aciuo vainillico Su 17 11 6u 68
Aciuo siiingico 1u S S Su S
Catequina 8 114 S1 11u 9S
vainillina 19 4u - - -
Siiingaluehiuo S 1S - 4u -
Epicatequina 8 29 SS S4u S1


Aunque este métouo piesenta uiveisas ventajas, también tiene
algunas limitaciones, sienuo piobablemente la más uestacable la
imposibiliuau ue conseguii que touos los analitos ensayauos oiiginen
ueiivauos fluoiescentes. Solo los áciuos hiuioxibenzoicos, catequinas y
Biscusion ue los iesultauos
21S
aluehiuos fenolicos han piesentauo fluoiescencia, sienuo imposible bajo las
conuiciones expeiimentales fijauas en el métouo que los áciuos
hiuioxicinámicos, flavonoiues y estilbenos ensayauos foimaian ueiivauos
fluoiescentes.

Beteiminacion ue compuestos flavonoiues en zumos ue naianja meuiante
luminiscencia sensibilizaua ue teibio.

El métouo piopuesto peimite ueteiminai cinco flavonoiues en
zumos ue naianja iealizanuo su sepaiacion ciomatiogiáfica y posteiioi
ueiivatizacion meuiante la foimacion simultanea ue quelatos con los
iones aluminio(III) y teibio(III) paia obtenei luminiscencia sensibilizaua.
La intensiuau ue estos quelatos es mayoi que la obteniua utilizanuo solo
teibio(III), lo que pueue atiibuiise a que el aluminio(III) oiigina un efecto
ue co-luminiscencia |Sj.

Entie las innovaciones que piesenta este métouo cabe uestacai que
es la piimeia vez que se uesciibe el uso ue una columna monolitica paia
la sepaiacion ue estos compuestos en zumos ue naianja y la foimacion
simultanea ue quelatos ue uos iones metálicos paia obtenei luminiscencia
sensibilizaua. El apoite ue estas innovaciones iepeicute uiiectamente en
las ventajas uel métouo: 1) no se iequieie una etapa pievia ue
tiatamiento ue muestia, 2) el volumen ue inyeccion ue muestia (2uuµL)
es supeiioi al ue otios métouos, y S) el sistema ue ueiivatizacion
uesaiiollauo peimite obtenei señales luminiscentes más intensas que las
obteniuas pieviamente utilizanuo solo teibio(III) como ieactivo
ueiivatizante |4j.

En la Tabla 2 se muestia la compaiacion ue los limites ue ueteccion
obteniuos con el métouo piopuesto con los uesciitos en otios métouos ya
estableciuos |6, 7j, uonue se pueue compiobai la excelente mejoia ue los
mismos. Asi, el limite ue ueteccion paia la naiingina es 12Su veces menoi
en el nuevo métouo que en el uesciito utilizanuo ueteccion fotométiica
|6j, y 1uuu veces menoi que con ueteccion electioquimica |7j. Paia el
kaempfeiol se ha obteniuo un limite ue ueteccion que es 12Su, Suu y 1uu
veces menoi que los uesciitos utilizanuo ueteccion electioquimica, masas
y fotométiica, iespectivamente |7j.

Capitulo S
216
Tabla 2. Compaiacion ue los L0Bs entie uifeientes métouos

Analito
Nétouo Piopuesto
LC-FB
|6j
|7j
LC-0v LC-NS LC-EB
L0B (ng mL
-1
) L0B (ng mL
-1
) L0B (ng mL
-1
)
Naiingina 1.u 12Su 2Su 1Su 1uuu
Bespeiiuina 2.7 1uuu Suu 1uu Suu
Queicetina 1.u 2Suu 1Su 2uu Suu
Naiingenina 1.S - - - -
Kaempfeiol u.8 - 8u 4uu 1uuu


Aunque este métouo piesenta uiveisos aspectos positivos, los
iesultauos pouiian habei siuo mejoies si se hubieia pouiuo extenuei a la
ueteiminacion ue un mayoi númeio ue compuestos fenolicos. También
hubieia siuo ueseable la utilizacion ue mayoies cauuales ue fase movil en la
sepaiacion ciomatogiáfica, peio el cauual se tuvo que auaptai a las
caiacteiisticas uel sistema ue ueiivatizacion que piesentaba pioblemas ue
piesion. No obstante, el tiempo necesaiio paia la sepaiacion es menoi que
el iequeiiuo utilizanuo las tiauicionales columnas ue fase ieveisa.


Beteiminacion ue flavonoiues en zumos ue naianja meuiante el uso ue
fluoiescencia ue laiga longituu ue onua.

Neuiante este métouo se han pouiuo ueteiminai seis ue los
flavonoiues más iepiesentativos piesentes en los zumos ue naianja
meuiante su sepaiacion ciomatogiáfica y posteiioi ueiivatizacion
utilizanuo ceiio(Iv) y el fluoiofoio ue laiga longituu ue onua violeta ue
ciesilo.

El uso ue fluoiofoios ue laiga longituu ue onua como ieactivos es
una opcion inteiesante paia mejoiai la selectiviuau uel análisis
fluoiimétiico y paia evitai o minimizai la etapa uel tiatamiento ue
muestia en el pioceso analitico. Estos fluoiofoios peimiten iealizai
meuiuas en una iegion uel espectio electiomagnético (>6uu nm) uonue la
absoicion o emision ue la matiiz ue la muestia es minima. Auemás, la
inteifeiencia Raman es en gian meuiua evitaua y la piobabiliuau ue
Biscusion ue los iesultauos
217
piocesos ue inhibicion no iauiantes se ieuuce uebiuo a la coita uuiacion
ue la fluoiescencia ue estos fluoiofoios.

Al igual que en los métouos anteiioies, se tiabaja con una columna
monolitica uisminuyenuo la uuiacion ue la sepaiacion, la cual se consigue
en unos 12 minutos, fiente a los SS minutos iequeiiuos en otios métouos
|8j. El análisis ue zumos iequieie fiecuentemente iecuiiii a piocesos ue
tiatamiento ue muestia como extiaccion, hiuiolisis, centiifugacion y¡o
filtiacion |6-7,9-12j. Sin embaigo, en el métouo piesentauo uestaca la
inexistencia uel tiatamiento ue muestia, salvo piocesos ue uilucion, lo
que uisminuye la complejiuau y uuiacion global uel pioceso analitico. La
compaiacion ue los limites ue ueteccion obteniuos fiente a los uesciitos
paia otios métouos, mayoiitaiiamente fotométiicos, piopuestos paia
ueteiminai estos compuestos (Tabla 3), peimite compiobai la notable
mejoia logiaua meuiante el nuevo métouo ya que en touos los casos se
consiguen limites ue ueteccion muy infeiioies. Esta uisminucion vaiia
entie 7uu veces menoi paia naiingina y 7 veces menoi paia naiingenina.


Tabla 3. Compaiacion ue los L0Bs entie uifeientes métouos.

Analito
Nétouo Piopuesto
LC-FB
|8j |6j
LC-0v
|7j
LC-0v LC-0v LC-NS
L0B (ng mL
-1
)
Rutina 1.S 4u - - -
Naiingina 1.8 Su 12Su 2Su 1Su
Bespeiiuina S.u - 1uuu Suu 1uu
Queicetina S.7 1uu 2Suu 1Su 2uu
Naiingenina 2.u 14 - - -
Kaempfeiol 1.u - - 8u 4uu


0na limitacion uel métouo piopuesto ha siuo la imposibiliuau ue
compatibilizai el sistema ue ueiivatizacion con cauuales mayoies ue fase
movil en la sepaiacion ciomatogiáfica. También hubieia siuo ueseable
ampliai el métouo a la ueteiminacion ue un mayoi númeio ue compuestos
fenolicos. Se ensayaion uiveisos áciuos fenolicos e hiuioxicinámicos como
los áciuos gálico, cafeico y p-coumaiico, peio no se consiguio una
sepaiacion ciomatogiáfica auecuaua ya que se eluian con el fiente ue
uisolvente.
Capitulo S
218

Compaiacion geneial ue los métouos uesaiiollauos

0na vez compaiauos inuiviuualmente caua uno ue los ties métouos
ciomatogiáficos piopuestos con otios métouos pieviamente uesciitos, se
iealiza a continuacion la compaiacion ue las caiacteiisticas piincipales ue
estos métouos entie si, ya que touos ellos implican sepaiacion,
ueiivatizacion y ueteccion fluoiimétiica ue analitos similaies. Paia
simplificai la compaiacion se han numeiauo los métouos según el sistema
ueiivatizante utilizauo, como se inuica a continuacion:

• Nétouo 1: teibio(III)
• Nétouo 2: aluminio(III) y teibio (III)
• Nétouo S: ceiio(Iv) y violeta ue ciesilo

A) Pioceso ue sepaiacion

En la Tabla 4 se compaian el númeio ue analitos, la uuiacion, el
númeio ue componentes ue la fase movil y el cauual utilizauo en caua caso.
Como pueue obseivaise, el númeio ue analitos sepaiauos en el métouo 1 es
muy supeiioi al ue los otios uos métouos, aunque solo 1u ue los 18 analitos
oiiginan iespuesta fluoiescente. En los métouos 2 y S se han sepaiauo los
mismos analitos peio incluyenuo en el métouo S a la iutina. Es ue uestacai
que la sepaiacion en este métouo se iealiza en la mitau ue tiempo que en el
métouo 2. Aunque los componentes ue la fase movil son los mismos en
ambos métouos (uisolucion iegulauoia ue áciuo acético (pB 4), acetonitiilo
y metanol), el giauiente ue elucion utilizauo en el métouo S uisminuye
notablemente la uuiacion uel pioceso ue sepaiacion. En lo iefeiente al
cauual ue fase movil, éste es bastante mayoi en el métouo 1.

Tabla 4. Compaiacion ue las caiacteiisticas ue la sepaiacion

Sepaiacion
Nétouo
1
Nétouo 2 Nétouo S
Nº analitos
Tiempo ue sepaiacion (min)
18
2S
S
2S
6
12

Nº uisolventes en la fase movil 2 S S
Cauual ue fase movil |mL min
-1
j 2 u.7 u.7

Biscusion ue los iesultauos
219
B) Sistema ue ueiivatizacion

En la Tabla 5 se muestian los uistintos componentes uel sistema
ueiivatizacion utilizauo en caua métouo, en el que se incluye en touos los
casos la piesencia ue agentes suifactantes. También se incluyen los
cauuales utilizauos, sienuo menoi en el métouo 2 y bastante similaies en
los otios uos métouos.

El métouo 1 es el más complejo ya que implica el uso ue un mayoi
númeio ue ieactivos en el sistema ue ueiivatizacion. El AEBT fue utilizauo
piincipalmente paia evitai la piecipitacion uel teibio(III), uebiuo al
elevauo pB utilizauo, aunque también se compiobo que mejoiaba la señal
uel áciuo gálico. El uso ue suifactantes se justifica piincipalmente poi su
efecto piotectoi en la luminiscencia ue los quelatos con teibio(III), aunque
el Tiiton X-1uu también aumento la señal uel áciuo gálico, mientias que el
SBS fue necesaiio paia conseguii aumentai la señal luminiscente uel
siiingaluehiuo, la cual fue muy baja en su ausencia.

En el métouo 2 no se iequiiio el uso ue AEBT ya que el pB utilizauo
fue bastante menoi (4.u) que en el métouo 1, poi lo que no existia iiesgo ue
que se piouujeia la piecipitacion uel teibio(III). Auemás, se compiobo que
su piesencia no mejoiaba las señales luminiscentes ue los analitos.
Tampoco fue necesaiio el uso ue T0P0 como agente sineigético peio, ue los
uistintos suifactantes ensayauos, el SBS mejoio notablemente las señales
analiticas a nivel ue concentiacion sub-micelai.

El sistema ueiivatizante utilizauo en el métouo S peimite la
iealizacion ue las meuiuas a mayoi longituu ue onua que en los métouos
anteiioies, uebiuo al uso uel fluoiofoio violeta ue ciesilo, lo que confieie al
métouo ue una mayoi selectiviuau fiente a la matiiz ue la muestia, como se
ha uiscutiuo anteiioimente. La obtencion ue picos ciomatogiáficos
negativos es el iesultauo ue la inhibicion que causan inuiiectamente los
analitos en la fluoiescencia uel fluoiofoio uebiuo a una combinacion ue
piocesos ieuox, áciuo-base e hiuiofobicos. Cabe uestacai que el cauual
utilizauo en este sistema es bastante mayoi que en el métouo 2, lo que es
atiibuible a que las cinéticas ue los piocesos implicauos en la
ueiivatizacion son más iápiuas que en los piocesos ue co-luminiscencia
Capitulo S
22u
que iequieie la foimacion ue los quelatos ue los analitos con aluminio(III) y
teibio(III).

Tabla 5. Compaiacion uel sistema ue ueiivatizacion

Beiivatizacion Nétouo 1 Nétouo 2 Nétouo S
Componentes empleauos
Tb(III)
AEBT
T0P0
TRIT0N X-1uu
Tb(III)
Al(III)
SBS
violeta ue ciesilo
CTAB
Ce(Iv)
Cauual ue uisolucion
ueiivatizante (mL min
-1
j
u.8 u.S u.9


C) Limites ue ueteccion y piecision

La Tabla 6 iecoge los limites ue ueteccion obteniuos paia los
analitos ueteiminauos en los ties métouos. Aunque no se pueue hacei una
compaiacion uiiecta ue los iesultauos obteniuos, ya que la mayoiia ue los
analitos uel métouo 1 no coinciuen con los ue los otios uos métouos, es ue
uestacai que en touos los casos se alcanzan limites ue ueteccion muy bajos,
a niveles ue ng mL
-1
, aunque éstos son en geneial más elevauos en el
métouo 1. Paia este métouo solo se han incluiuos los obteniuos meuiante
ueteccion fluoiimétiica, a excepcion ue los coiiesponuientes a iutina,
queicetina y kaempfeiol, obteniuos meuiante ueteccion fotométiica, ya que
éstos también están incluiuos en los otios uos métouos. Como pueue
obseivaise, los limites ue ueteccion paia estos analitos son bastante
menoies cuanuo se utiliza ueteccion fluoiimétiica.













Biscusion ue los iesultauos
221
Tabla 6. Compaiacion ue limites ue ueteccion

Nétouo 1 Nétouo 2 Nétouo S
Analito L0B (ng mL
-1
) L0B (ng mL
-1
) L0B (ng mL
-1
)
Aciuo gálico S - -
Aciuo piotocateuico 2 - -
Aciuo p-hiuioxibenzoico 1S - -
Aciuo salicilico 14 - -
Aciuo vainillico 17 - -
Aciuo siiingico S - -
Catequina 114 - -
vainillina 4u - -
Siiingaluehiuo 1S - -
Epicatequina 29 - -
Rutina 24* - 1.S
Naiingina - 1.u 1.8
Bespeiiuina - 2.7 S.u
Queicetina 6u* 1.u S.7
Naiingenina - 1.S 2.u
Kaempfeiol 18* u.8 1.u
*Beteccion fotométiica

Si se compaian los iesultauos ue los métouos 2 y S, uonue se han
utilizauo los mismos analitos, a excepcion ue la iutina, se obseiva que
piácticamente se alcanzan los mismos valoies ue los limites ue ueteccion,
aunque son ligeiamente más bajos en el métouo 2.

En lo iefeiente a la ue piecision ue los ties métouos, expiesaua
como %RSB, estos valoies oscilan entie u,u8-6,4%, 1,S-4,7% y 2,8-6,2%
paia los métouos 1, 2 y S, iespectivamente. Aunque en el métouo 1 se
alcanzan valoies bastante bajos en algunos casos, en geneial no existen
uifeiencias muy significativas entie ellos.

Como iesumen ue la compaiacion ue los ties métouos
ciomatogiáficos uesciitos en esta Nemoiia, y consiueianuo que caua uno
ue ellos tiene sus coiiesponuientes ventajas y limitaciones, pueuen
ueuuciise las conclusiones siguientes:

1) Los bajos limites ue ueteccion obteniuos en los ties casos peimiten
el análisis uiiecto ue las muestias meuiante su auecuaua uilucion.
2) El métouo 1 es el que piesenta mayoi iesolucion uebiuo al elevauo
númeio ue analitos sepaiauos, aunque el sistema ue ueiivatizacion
es más complejo y los limites ue ueteccion son más elevauos.
Capitulo S
222
S) Los mejoies limites ue ueteccion se obtienen con el métouo 2, peio
la sepaiacion es más lenta que en el métouo S.
4) El métouo S es el que utiliza piobablemente un sistema ue
ueiivatizacion más oiiginal y que peimite conseguii una sepaiacion
más iápiua.


Nétouo quimiométiico ue clasificacion

Como consecuencia ue la infoimacion analitica obteniua en el
métouo piopuesto paia la ueteiminacion compuestos fenolicos en vinos
meuiante luminiscencia sensibilizaua ue teibio, se ha uesaiiollauo un
métouo ue clasificacion ue vinos meuiante técnicas quimiométiicas basauo
en el conteniuo en compuestos fenolicos, vaiieuau ue vino y oiigen
geogiáfico.

La matiiz ue uatos constiuiua con la concentiacion ue caua uno ue
los compuestos fenolicos hallauos en caua muestia ha siuo sometiua a
uifeientes algoiitmos matemáticos paia caiacteiizai y clasificai
auecuauamente las muestias ue vino. Se ha hecho uso ue métouos
supeivisauos (análisis uisciiminante lineal) y no supeivisauos (análisis uel
factoi) ue pautas.

Se han uesciito uiveisos métouos ue clasificacion ue vinos. Como
ejemplos caben citai uos métouos que iequieien un númeio elevauo ue
compuestos fenolicos, 19 o más, y que utilizan el oiigen geogiáfico y la
vaiieuau ue uva |1Sj o el año ue cosecha |14j, obtenienuo en ambos casos
un 9S% ue efectiviuau en la clasificacion ue vinos. En el métouo piopuesto
se ha uemostiauo que, aunque el métouo ue análisis apoita infoimacion
sobie 18 analitos utilizanuo ueteccion fotométiica y sobie 1u ue ellos
utilizanuo ueteccion fluoiimétiica, solo con esta última infoimacion se
pueue establecei un pioceuimiento quimiométiico que alcanza un 1uu% ue
efectiviuau paia la clasificacion ue las muestias.




Biscusion ue los iesultauos
22S
2. Metodologías automáticas no cromatográficas basadas en el uso
de la técnica de flujo detenido y nanopartículas

En este apaitauo se comentan y uiscuten los iesultauos obteniuos
en el establecimiento ue uos métouos paia la ueteiminacion ue
compuestos fenolicos y otios antioxiuantes en muestias ue alimentos. En
ambos métouos se ha utilizauo la técnica ue flujo ueteniuo paia
automatizai la mezcla ue los ieactantes y la obtencion ue iesultauos
analiticos. El objetivo básico ue este estuuio ha siuo el uesaiiollo ue
nuevas metouologias ue análisis intiouucienuo en el sistema analitico el
uso ue nanomateiiales y¡o enzimas que peimitan mejoiai las
piopieuaues analiticas ue los métouos ya existentes. Los métouos
estableciuos utilizan uos paiámetios cinéticos, velociuau inicial ue la
ieaccion y peiiouo ue inuuccion, paia la cuantificacion ue los analitos
meuiante meuiuas ue iauiacion uispeisaua ue iesonancia en el piimei
caso y ue fluoiescencia ue laiga longituu ue onua en el segunuo. Asi, se
han uesaiiollauo:

• 0n métouo paia la ueteiminacion ue antioxiuantes meuiante
meuiua uiiecta ue la foimacion ue nanopaiticulas ue oio
(AuNPs) utilizanuo la iauiacion uispeisaua ue iesonancia como
sistema ue ueteccion.
• 0n métouo enzimático-fluoiimétiico paia la ueteiminacion ue
polifenoles basauo en la inhibicion tempoial que ejeicen estos
compuestos en la oxiuacion ue un fluoiofoio ue laiga longituu
ue onua.

Beteiminacion ue auitivos antioxiuantes en muestias alimentaiias
meuiante la meuiua uiiecta ue la foimacion ue nanopaiticulas ue oio poi
uispeision ue la iauiacion.

La ueteiminacion ue antioxiuantes alimentaiios se iealiza
geneialmente meuiante ciomatogiafia ue liquiuos y usanuo fotometiia o
espectiometiia ue masas como sistemas ue ueteccion |1S,16j. También se
han uesciito métouos basauos en ciomatogiafia ue gases con uetectoi ue
ionizacion ue llama o espectiometiia ue masas, y en electiofoiesis capilai
con ueteccion fotométiica o electioquimica |1Sj. En geneial, los limites ue
ueteccion alcanzauos en estos métouos se encuentian a niveles ue µmol L
-1
.
Capitulo S
224
Aunque el métouo piopuesto no incluye la sepaiacion ue
antioxiuantes y, poi tanto, no peimite su cuantificacion inuiviuual,
constituye una alteinativa oiiginal y iápiua como métouo ue scieening paia
uetectai la piesencia ue antioxiuantes natuiales y¡o sintéticos en alimentos.
Se uemuestia poi piimeia vez la posibiliuau ue utilizai con fines analiticos
la foimacion ue AuNPs inuuciua poi antioxiuantes, usanuo la velociuau
inicial uel pioceso, obteniua miuienuo la vaiiacion ue la uispeision ue la
iauiacion ue iesonancia con el tiempo, como paiámetio analitico. La
meuiua ue este paiámetio se iealiza en unos cinco segunuos, sin necesiuau
ue utilizai ninguna etapa pievia ue incubacion. Es ue uestacai la necesiuau
ue llevai a cabo el pioceso en piesencia uel suifactante cationico CTAB, lo
que se atiibuye a que la foimacion ue un pai ionico con el anion AuCl
4
-

favoiece la ieaccion ieuox.

Los limites ue ueteccion alcanzauos vaiian entie u.uu7 µmol L
-1

paia galato ue uouecilo y u.uS µmol L
-1
paia áciuo ascoibico. Al compaiai
estos valoies con los limites obteniuos poi otios métouos, con uiveisos
sistemas ue ueteccion |1Sj, se pueue establecei que el métouo uesaiiollauo
mejoia los valoies uesciitos. Poi ejemplo, en el caso ue los galato ue
piopilo y octilo, los limites ue ueteccion uesciitos son u.29 y 2.S µmol L
-1
,
iespectivamente, mientias que paia el métouo piopuesto los valoies
obteniuos paia estos analitos son u.u1 y u.uu8 µmol L
-1
, iespectivamente.

El métouo se ha aplicauo satisfactoiiamente al análisis ue vaiios
alimentos, obtenienuo iesultauos similaies a los obteniuos meuiante
ciomatogiafia ue liquiuos y, en el caso uel áciuo ascoibico, meuiante
valoiacion ieuox.


Beteiminacion uel conteniuo ue polifenoles en bebiuas empleanuo laccasa,
AuNPs y fluoiimetiia ue laiga longituu ue onua.

Como se ha inuicauo en la intiouuccion ue esta Tesis, el uesaiiollo
ue métouos paia ueteiminai el conteniuo ue polifenoles totales en
alimentos continua sienuo un áiea ue investigacion ue gian inteiés. Los
métouos más empleauos son el métouo fotométiico oficial ue la A0AC
(Folin-Ciocalteu) |17j y métouos basauos en biosensoies ampeiométiicos
|18,19j. El métouo piopuesto ofiece una alteinativa innovauoia meuiante
Biscusion ue los iesultauos
22S
cuatio elementos que lo uifeiencian ue los métouos anteiioimente
uesciitos:

1) 0tiliza fluoiimetiia ue laiga longituu ue onua como sistema ue
ueteccion lo que, como pieviamente se ha uiscutiuo, confieie al
métouo cieita selectiviuau paia evitai posibles inteifeiencias ue la
matiiz ue la muestia.
2) En lugai ue un ieactivo que actúa fiente a cualquiei ieuuctoi
piesente en la muestia, como ocuiie con el ieactivo Folin-Ciocalteu,
utiliza la enzima laccasa, que ofiece un mayoi giauo ue selectiviuau
paia la oxiuacion ue polifenoles.
S) El métouo se basa en la inhibicion tempoial que causan los
polifenoles en la oxiuacion uel fluoiofoio veiue ue inuocianina (i
ex

764 nm, i
em
8u6 nm), utilizauo un mouulo ue flujo ueteniuo paia
automatizai la mezcla ue los ieactantes y la meuiua uel peiiouo ue
inuuccion obteniuo, piopoicional a la concentiacion ue analito.
4) Incluye la utilizacion ue AuNPs caigauas positivamente, las cuales
inteiaccionan con la enzima y uisminuyen su activiuau,
consiguienuo mejoiai los limites ue ueteccion obteniuos.

Estos elementos han peimitiuo el uesaiiollo ue un métouo que
piesenta limites ue ueteccion bastante menoies que los obteniuos en la
mayoiia ue los métouos que emplean biosensoies ampeiométiicos, como
muestia la Tabla 7.

Tabla 7. Compaiacion ue los L0Bs entie uifeientes métouos

Analito
Nétouo
piopuesto
FLB
|18j |19j |2uj
Biosensoi Biosensoi
Biosensoi
L0B
(µmol L
-1
)
L0B
(µmol L
-1
)
L0B
(µmol mL
-1
)
L0B
(µmol mL
-1
)
Catecol u.u1 1u -
-
Biuioquinona u.u1 - -
-
Biuioxihiuioquinona u.uS - - -
Aciuo gálico u.u4 S8u u.41 17.u1
Piiogalol u.u4 - - -

0na limitacion uel métouo, al igual que ocuiie en los otios métouos
paia la ueteiminacion ue polifenoles totales, es que solo se obtiene una
Capitulo S
226
estimacion apioximaua ya que los iesultauos están iefeiiuos a un polifenol
que se utiliza como mouelo, geneialmente áciuo gálico. 0tio aspecto
ciiticable es el uso ue la enzima en meuio homogéneo, a uifeiencia ue los
biosensoies enzimáticos que implican su inmovilizacion, lo que posibilita
su ieutilizacion. No obstante, la cantiuau ue enzima utilizaua en caua
meuiua es extiaoiuinaiiamente baja, uebiuo a la elevaua sensibiliuau uel
métouo.

Biscussion of the iesults
227
Introduction

The iesults obtaineu thioughout this Boctoial Thesis aie uiscusseu
in this chaptei, incluuing the stiengths anu weakness of the methouologies
uevelopeu. Also, a compaiison of theii main featuies with those obtaineu
foi othei methous uesciibeu foi the same puipose is maue.

This chaptei is uiviueu in two sections. In the fiist one, the iesults
obtaineu fiom the methouologies baseu on chiomatogiaphic sepaiation,
ueiivatization, anu fluoiescence uetection aie uiscusseu anu, in the seconu
one, the iesults fiom the automateu non-chiomatogiaphic methouologies
pioposeu by the use of the stoppeu-flow technique aie uiscusseu.


1. Methodologies based on chromatographic separation, post-
column derivatization and fluorescence detection

This section ueals with the uiffeient chiomatogiaphic methous
uevelopeu foi the ueteimination of phenolic compounus in foou samples.
The aim of these methous was the establishment of new methouologies
which impiove the basic (piecision, selectivity anu sensitivity) anu the
complementaiy (speeu, cost anu peisonal factois) analytical piopeities of
the existing methous, so that the new pioposeu methous coulu be a ieal
alteinative foi the ueteimination of these compounus, which aie of gieat
inteiest in foou quality contiol.

Liquiu chiomatogiaphy as sepaiation technique anu luminescence as
uetection system have been useu to achieve the pioposeu objectives.
Bowevei, the innovation of the pioposeu methouology lies in the seveial
post-column ueiivatization systems useu in each of them to pioviue the
analytes (with no native fluoiescence) with luminescent piopeities. Thus,
the following methous have been uevelopeu:

• An analytical methou that involves a post-column
ueiivatization system baseu on the foimation of luminescent
chelates of the analytes with teibium(III) at a slightly basic
meuium, in the piesence of T0P0 anu EBTA.

Capitulo S
228
• An analytical methou that involves a post-column
ueiivatization system baseu on the capability of flavonoius to
foim stable chelates with aluminum(III) anu teibium(III) in
the piesence of a sub-micellai meuium pioviueu by SBS.

• An analytical methou baseu in a ueiivatization system in
which the analytes inteiact with ceiium(Iv) anu a long
wavelength fluoiophoi.


All the methouologies uevelopeu in this section shaie some
common elements that pioviue seveial auvantages ovei othei analytical
methous pieviously establisheu.

Fiist, the chiomatogiaphic column useu in the thiee pioposeu
methous is a monolithic C
18
column, in contiast with the ieveiseu-phase
columns packeu with silica miciopaiticles that aie most fiequently useu foi
the sepaiation of phenolic compounus. The use of this kinu of columns
pioviues the possibility of opeiate at highei flow iates with lowei back-
piessuie. Thus, they allow the analysis of samples using high sample
volumes, achieving a bettei analytic signal in foim of high anu naiiow
chiomatogiaphic peaks. Anothei auvantage pioviueu by this type of
columns is that they allow the analysis of samples using uiiect injection oi
low sample uilutions, because the cleaning anu iegeneiation of the column
can be uone at a high speeu.

Seconuly, a spectiofluoiimetei uetectoi is useu in all instances to
measuie the analytical signals at long oi ielative long wavelength, using an
appiopiiate fluoiophoi oi the luminescent chelates foimation of the
analytes with teibium(III), iespectively. The uetection in this pait of the
spectium minimizes potential inteifeiences fiom backgiounu sample
matiix as they tenu to emit at shoitei wavelengths. In auuition, the
selectivity offeieu by sensitizeu luminescence facilitates the iuentification
of some compounus that cannot be easily iuentifieu using photometiic
uetection.

Below, the iesults foi each of the methouologies uevelopeu aie
uiscusseu with moie uetail.
Biscussion of the iesults
229
Beteimination of phenolic compounus in wine by teibium-sensitizeu
luminescence

This methou allows the simultaneous ueteimination of 18 uiffeient
phenolic compounus, incluuing hyuioxybenzoic anu hyuioxycinnamic
acius, catechins, phenolic aluehyues, flavonols anu stilbenes, using
photometiic anu fluoiimetiic uetection. The methou is baseu on the
chiomatogiaphic sepaiation anu post-column ueiivatization of the analytes
to tiansfoim them into fluoiescent ueiivatives. The ueiivatization system
is baseu on the foimation of soluble anu stable chelates of the analyte-
liganus with teibium(III), in which the liganu is exciteu anu then the
absoibeu eneigy is tiansfeiieu to the lanthaniue, which finally emits
luminescence.

The auvantages of the methou piesenteu aie the following:
• Those ueiiveu fiom the use of a C
18
monolitic column, which
have uiscusseu eailiei.
• The ielatively long emission wavelength of teibium(III)
chelates can minimize existing inteifeiences in the sample
matiix, which typically emit at lowei wavelength.
Fuitheimoie, the selectivity of the sensitizeu luminescence
can facilitate the iuentification of some compounus that aie
not be easily uetecteu by photometiy.
• The only tieatment neeueu is sample uilution.
• This is the fiist time that aiomatic aluehyues have been
ueteimineu using teibium-sensitizeu luminescence.

These featuies have alloweu the chiomatogiaphic sepaiation in less
time than that uesciibeu in othei methous foi these phenolic compounus.
Fifteen phenolic compounus in wines samples have been sepaiateu in just
2S minutes. In contiast, othei methous |1-2j that use packeu columns
achieve the sepaiation of 14 to 2S phenols in 6u minutes, oi S2 compounus
in 9u minutes. Betection limits have been also impioveu, as shown in Table
1, in which a compaiison with othei methous pieviously uesciibeu |2-Sj is
incluueu. The uetection limits achieveu using fluoiimetiic uetection aie
lowei than those obtaineu using photometiic uetection in the 4u% of the
cases, which shows that sensitizeu luminescence helps not only to inciease
Capitulo S
2Su
the selectivity but also the sensitivity. Compaiing the pioposeu methou
with otheis, it can be concluueu that both photometiic anu fluoiimetiic
uetection limits aie impioveu in most cases, uemonstiating the utility of
the methou uevelopeu.

Table 1. Compaiison of L0Bs between uiffeient methous

Pioposeu Nethou |2j |Sj
Photometiy Fluoiimetiy Photometiy Photometiy Fluoiimetiy
Analyte L0B
(ng mL
-1
)
L0B
(ng mL
-1
)
L0B
(ng mL
-1
)
L0B
(ng mL
-1
)
L0B
(ng mL
-1
)
uallic aciu S S 14 6u -
Piotocatechuic
aciu
S6 2 18 12u -
p-hyuioxy benzoic
aciu
22 1S - - -
Salicylic aciu 14 14 - - -
vanillic aciu Su 17 11 6u 68
Syiingic aciu 1u S S Su S
Catechin 8 114 S1 11u 9S
vanillin 19 4u - - -
Syiingaluehyue S 1S - 4u -
Epicatechin 8 29 SS S4u S1

Although this methou has seveial auvantages, it has also some
limitations, being piobably the most iemaikable the impossibility to get
fluoiescent ueiivatives of all the analytes. 0nly hyuioxybenzoic acius,
catechins anu phenolic aluehyues have piesenteu fluoiescence, but it was
impossible to obtain fluoiescence signals fiom hyuioxycinnamic acius,
flavonoius anu stilbenes unuei the expeiimental conuitions set in the
methou.

Beteimination of flavonoius compounus in oiange juice using teibium
sensitizeu luminescence

The pioposeu methou can iuentify five flavonoius in oiange juices
by theii chiomatogiaphic sepaiation anu ueiivatization, which is baseu on
the capability of flavonoius to foim stable chelates with aluminum(III) anu
teibium(III), anu the measuiement of teibium sensitizeu luminescence.
The luminescence intensity of these chelates is highei than that obtaineu
using only teibium (III), which can be asciibeu to a co-luminescence effect
|Sj.

Biscussion of the iesults
2S1
The two main innovations of this methou aie the use of a monolithic
column foi the sepaiation of these compounus in oiange juices anu the
simultaneous foimation of chelates using two uiffeient metal ions to obtain
sensitizeu luminescence. These innovations aie uiiectly ielateu with the
benefits of the methou: 1) sample pie-tieatment is not iequiieu, 2) the
sample injection volume (2uu µL) is highei than that useu in othei
methouologies, anu S) the luminescence signals obtaineu by the pioposeu
ueiivatization system aie moie intense than those obtaineu using only
teibium(III) as the ueiivatizing ieagent |4j.

Table 2 shows a compaiison between the uetection limits achieveu
with the pioposeu methou anu those uesciibeu in othei existing methous
|6, 7j. As can be seen, an excellent impiovement on the uetection limits is
obtaineu. Foi instance, the naiingin uetection limit is 12Su anu 1uuu times
lowei than those iepoiteu using photometiic |6j anu electiochemical (EB)
uetection |7j, iespectively. Foi kaempfeiol, the uetection limit is 12Su, Suu
anu 1uu times lowei than those obtaineu in the methous uesciibeu using
electiochemical, mass spectiometiy anu photometiic uetection,
iespectively |7j.

Table 2. Compaiison of L0Bs between uiffeient methous

Analyte
Pioposeu Nethou
LC-FB
|6j
LC-0v
|7j
LC-0v LC-NS LC-EB
L0B (ng mL
-1
) L0B (ng mL
-1
) L0B (ng mL
-1
)
Naiingin 1.u 12Su 2Su 1Su 1uuu
Bespeiiuin 2.7 1uuu Suu 1uu Suu
Queicetin 1.u 2Suu 1Su 2uu Suu
Naiingenin 1.S - - - -
Kaempfeiol u.8 - 8u 4uu 1uuu


Although this methou has seveial positive aspects, it also shows two
main uiawbacks: 1) the numbei of phenolic compounus ueteimineu is
ielatively low, anu 2) A highei flow iate of mobile phase in the
chiomatogiaphic sepaiation woulu have been uesiiable, but the flow hau to
be auapteu to the chaiacteiistics of the ueiivatization system to avoiu
piessuie pioblems. Bowevei, the time iequiieu foi the sepaiation is lowei
than that iepoiteu in othei methous, which involve the use of tiauitional
ieveiseu phase columns.
Capitulo S
2S2
Beteimination of flavonoius in oiange juice using long wavelength
fluoiescence

A new methou foi the ueteimination of six of the most
iepiesentative flavonoius piesent in oiange juice has been uevelopeu. This
methou involves a chiomatogiaphic sepaiation anu a post-column
ueiivatization system using ceiium(Iv) anu the long wavelength fluoiophoi
ciesyl violet.

The use of long wavelength fluoiophoies as analytical ieagents is an
inteiesting option to impiove the selectivity of fluoiimetiic analysis anu
avoiu oi minimize the sample tieatment step of the analytical piocess.
These fluoiophoies allow measuiements in a iegion of the electiomagnetic
spectium (>6uunm) in which the potential absoiption oi emission
associateu to sample matiix is minimizeu. Also, Raman inteifeience is
gieatly uiminisheu anu the piobability of non-iauiative quenching
piocesses is uecieaseu uue to the usually shoit fluoiescence lifetime of
these fluoiophoies.

As in pievious methous, the use of a monolithic column uecieases
the length of the sepaiation, which is achieveu in about 12 minutes, in
contiast with the SS minutes iequiieu in othei methous |8j. }uice analysis
often iequiies sample tieatment piocesses such as extiaction, hyuiolysis,
centiifugation anu¡oi filtiation |6-7,9-12j. Bowevei, these piocesses aie
avoiueu in the pioposeu methou, except a uilution step, so the complexity
anu the oveiall length of the analytical piocesses aie uiminisheu.

Compaiing the uetection limits obtaineu in the pioposeu methou to
those uesciibeu in othei methous, mainly photometiic methous, uesciibeu
foi the ueteimination of these compounus (Table 3), it can be checkeu the
iemaikable impiovement achieveu in all instances. Thus, the ueciease
obtaineu foi the uetection limits ianges between 7uu anu 7 times lowei foi
naiingin anu naiingenin, iespectively.





Biscussion of the iesults
2SS
Table 3. Compaiison of L0Bs between uiffeient methous


Analyte

Pioposeu Nethou
LC-FB
|8j |6j
LC-0v
|7j
LC-0v LC-0v LC-NS
L0B (ng mL
-1
)
Rutin 1.S 4u - - -
Naiingin 1.8 Su 12Su 2Su 1Su
Bespeiiuin S.u - 1uuu Suu 1uu
Queicetin S.7 1uu 2Suu 1Su 2uu
Naiingenin 2.u 14 - - -
Kaempfeiol 1.u - - 8u 4uu


In spite of the positive aspects of the pioposeu methou, the
ueteimination of a highei numbei of phenolic compounus woulu be
uesiiable. Bowevei, seveial phenolic anu hyuioxycinnamic acius, such as
gallic, caffeic anu p-coumaiic acius weie testeu but the chiomatogiaphic
sepaiation was not possible as they eluteu with the solvent fiont. In
auuition, it woulu have been uesiiable the use of highei flow iates of
mobile phase in the chiomatogiaphic sepaiation, but the flow hau to be
auapteu to the chaiacteiistics of the ueiivatization system to avoiu
piessuie pioblems.

ueneial compaiison of the methous uevelopeu

Aftei the inuiviuual compaiison of the thiee pioposeu
chiomatogiaphic methous with othei methous pieviously uesciibeu, now
the uiiect compaiison of the main featuies of the new methous is
piesenteu, as all of them involve the use of chiomatogiaphic sepaiation,
ueiivatization anu fluoiimetiic uetection of similai analytes. To simplify
the compaiison, the methous have been numbeieu accoiuing to the
ueiivatizing system, as follows:

• Nethou 1: teibium(III)
• Nethou 2: aluminium(III) anu teibium(III)
• Nethou S: ceiium(Iv) anu ciesyl violet




Capitulo S
2S4
A) Sepaiation piocess

The numbei of analytes, the length of each sepaiation, the numbei
of mobile phase components anu its flow iate aie compaieu in Table 4. As
shown, the numbei of analytes sepaiateu in methou 1 is much highei than
in the othei two methous, although only 1u of the 18 analytes oiiginate a
fluoiescent iesponse. In methous 2 anu S the same analytes have been
sepaiateu, but incluuing iutin in methou S. It is iemaikable that the
sepaiation in methou S is peifoimeu in half time than in methou 2.
Although the components of the mobile phase aie the same in both
methous |acetic buffei solution (pB 4), acetonitiile anu methanolj, the
elution giauient useu in methou S significantly uecieaseu the length of the
sepaiation piocess. Regaiuing the mobile phase flow, it is consiueiably
highei in methou 1.

Table 4. Compaiison of the sepaiation chaiacteiistics

Sepaiation Nethou 1 Nethou 2 Nethou S
Analytes
Sepaiation time (min)
18
2S
S
2S
6
12

Nobile phase components 2 S S
Nobile phase flow |mL min
-1
j 2 u.7 u.7



B) Beiivatization system

Table 5 shows the uiffeient components of the ueiivatization
system useu in each methou in which the use of suifactants is necessaiy in
all instances. The flows useu aie also incluueu, being lowei in methou 2 anu
quite similai in the othei two methous.

Nethou 1 is the most complex because it involves the use of a
gieatei numbei of ieagents in the ueiivatization system. EBTA was mainly
useu to pievent teibium(III) piecipitation uue to the high pB useu, but it
impioves also the signal of gallic aciu. The use of suifactants is mainly
justifieu by its piotective effect on the luminescence of teibium(III)
chelates. Tiiton X-1uu also incieaseu the signal of gallic aciu, while SBS was
Biscussion of the iesults
2SS
necessaiy to achieve an inciease in the luminescence signal of
syiingaluehyue, which was veiy low in its absence.

The use of EBTA was not iequiieu in methou 2, as the pB useu was
significantly lowei (4.u) than in methou 1, so theie is no iisk about
teibium(III) piecipitation. In auuition, it was founu that its piesence uiu
not impiove the luminescent signal of the analytes anu, also, the use of
T0P0 as syneigetic agent was no necessaiy. Bowevei, the assay of the
potential effect of uiffeient suifactants on the system showeu that SBS
gieatly impioveu the analytical signals at a sub-micellai concentiation.

The ueiivatizing system useu in methou S allows the measuiement
at a longei wavelength that in the pievious methous because of the use of
the fluoiophoi ciesyl violet enuows the methou with a bettei selectivity, as
it has been uiscusseu befoie. The negative chiomatogiaphic peaks aie the
iesults of the inuiiect inhibition in the ciesyl violet fluoiescence by the
analytes uue to a combination of ieuox, aciu-base anu hyuiophobic
piocesses. The flow useu in this system is highei than in methou 2, which is
asciibeu to the kinetics of the piocess involveu in the ueiivatization, which
aie fastei than those involveu in the co-luminescence piocesses iequiiing
the chelates foimation with aluminium(III) anu teibium(III).

Table 5. Compaiison of the ueiivatization systems

Beiivatization Nethou 1 Nethou 2 Nethou S
Components
Tb(III)
AEBT
T0P0
TRIT0N X-1uu
Tb(III)
Al(III)
SBS
Ciesyl violet
CTAB
Ce(Iv)
Beiivatizing solution flow
(mL min
-1
j
u.8 u.S u.9


C) Betection limits anu piecision

Table 6 shows the uetection limits obtaineu in the thiee methous. A
uiiect compaiison of all the iesults obtaineu cannot be maue because most
of the analytes in methou 1 uo not coinciue with those in the othei
methous. Bowevei, it can be seen that the uetection limits aie veiy low in
all instances, ieaching levels of ng mL
-1
, although these values aie geneially
Capitulo S
2S6
highei foi methou 1. Foi this methou only those obtaineu by fluoiimetiic
uetection have been incluueu, with the exception foi iutin, queicetin anu
kaempfeiol, obtaineu with photometiic uetection. As shown, the uetection
limits foi these analytes aie significantly lowei using fluoiimetiic
uetection.


Table 6. Compaiison of uetection limits

Nethou 1 Nethou 2 Nethou S
Analito L0B (ng mL
-1
) L0B (ng mL
-1
) L0B (ng mL
-1
)
uallic aciu S - -
Piotocatechuic aciu 2 - -
p-Byuioxybenzoic aciu 1S - -
Salicylic aciu 14 - -
vanillic aciu 17 - -
Syiingic aciu S - -
Catechin 114 - -
vanillin 4u - -
Syiingaluehyue 1S - -
Epicatechin 29 - -
Rutin 24* - 1.S
Naiingin - 1.u 1.8
Bespeiiuin - 2.7 S.u
Queicetin 6u* 1.u S.7
Naiingenin - 1.S 2.u
Kaempfeiol 18* u.8 1.u
* Photometiic uetection


Compaiing the iesults of methous 2 anu S, in which the same
analytes aie involveu, except foi iutin, the uetection limit levels ieacheu
aie veiy similai, although it seems to be lowei in methou 2.

Regaiuing the piecision of the thiee methous, expiesseu as % RSB,
these values iangeu between u.u8 - 6.4%, 1.S - 4.7 % anu 2.8 - 6.2% foi
methous 1, 2 anu S iespectively. Although methou 1 ieaches veiy low
values in some cases, theie aie no significant uiffeiences between them.

To summaiize this compaiison between methous anu consiueiing
that each one has its auvantages anu limitations, the following conclusions
can be establisheu:
Biscussion of the iesults
2S7
1) The low uetection limits obtaineu in the thiee methous
allow the uiiect sample analysis by the appiopiiate
uilution.
2) Nethou 1 achieves the highest iesolution uue the high
numbei of analytes sepaiateu, although the ueiivatization
system is moie complex anu its uetection limits aie highei.
S) Nethou 2 gives the best uetection limits, but the sepaiation
is slowei than methou S.
4) Nethou S involves piobably the most oiiginal ueiivatization
system anu allows a fastei sepaiation.


Chemometiic classification methou

As a iesult of the analytical infoimation obtaineu fiom the pioposeu
methou foi the ueteimination of phenolic compounus in wine by teibium-
sensitizeu luminescence, a chemometiic classification methou has been
uevelopeu. This methou involves the use of chemometiic techniques to
classify wines anu it is baseu on the phenolic content, vaiiety anu
geogiaphical oiigin.

The pattein uata matiix, constiucteu by the concentiation of each
analyte piesent in each wine sample, was subjecteu to uiffeient algoiithms
in oiuei to classify anu chaiacteiize the wine samples auequately.
Supeiviseu (lineai uisciiminant analysis) anu un-supeiviseu (factoi
analysis) pattein iecognition methous have been useu.

Seveial classification methous of wines have been uesciibeu. Foi
example, theie aie two methous that iequiie a high numbei of phenolic
compounus, 19 oi moie, anu use the geogiaphical oiigin anu the giape
vaiiety |1Sj oi the vintage yeai |14j as classification pattein, obtaining in
both cases a 9S% effective classification. The infoimation pioviueu by the
pioposeu methou is obtaineu fiom 18 analytes, using photometiic
uetection, anu only 1u of them give fluoiimetiic iesults. Bowevei, a
chemometiic pioceuuie foi the classification of the samples that ieaches
1uu% effectiveness can be establisheu using only the fluoiimetiic
infoimation.

Capitulo S
2S8
2. Automatic non-chromatographic methodologies based in the
used of stopped-flow technique and nanoparticles.

The iesults obtaineu in two methous foi the ueteimination of
phenolic compounus anu othei antioxiuants in foou samples aie uiscusseu
in this section. The stoppeu-flow technique has been useu in both methous
to automate the mixing of the ieactants anu to obtain analytical iesults. The
aim of this stuuy has been the uevelopment of new analysis methous with
the use of nanomateiials anu¡oi enzymes, which allow the impiovement of
the analytical piopeities of the existing methous. Two kinetic paiameteis,
initial ieaction iate anu inuuction peiiou, have been useu in these methous
to quantify the analytes, anu iesonance light scatteiing anu long
wavelength fluoiescence have been useu as the uetection systems. Thus, it
has been uevelopeu:

• A methou foi antioxiuant ueteimination by the uiiect
measuiement of golu nanopaiticles (AuNPs) foimation using
iesonance light scatteiing as uetection system.
• An enzymatic-fluoiimetiic methou foi the ueteimination of
polyphenols baseu on the tempoiaiy inhibition causeu by
these compounus on the oxiuation of a long wavelength
fluoiophoi.


Beteimination of antioxiuant auuitives in foou stuffs by uiiect
measuiement of golu nanopaiticles foimation using iesonance light
scatteiing uetection.

The ueteimination of foou antioxiuants is mainly caiiieu out by
using liquiu chiomatogiaphy with photometiy oi mass spectiometiy as
uetection systems |1S,16j. Nethous baseu on gas chiomatogiaphy with
flame ionization uetectoi oi mass spectiometiy, anu capillaiy
electiophoiesis with photometiic oi electiochemical uetection have also
been uesciibeu |1Sj. The limits of uetection iepoiteu foi most of these
methous aie at level of µmol L
-1
.

The pioposeu methou uoes not incluue the sepaiation of the
antioxiuants so theii inuiviuual quantification is not alloweu. Bowevei, it
Biscussion of the iesults
2S9
can be seen as an oiiginal anu quick alteinative as a scieening methou to
uetect the piesence of natuial anu¡oi synthetic antioxiuants in foou. The
usefulness of kinetic methouology foi monitoiing the foimation of AuNPs
inuuceu by antioxiuant compounus is shown foi the fiist time. The initial
iate of the piocess, which is obtaineu by measuiing the vaiiation of the
iesonance light scatteiing signal with time, is useu as the analytical
paiametei. The measuiement of this paiametei takes about five seconus
anu a pievious stage of incubation is not neeueu. It is iemaikable the neeu
of the cationic suifactant CTAB to caiiy out the piocess because the
foimation of an ionic paii between AuCl
4
-
anu the cationic suifactant is
necessaiy, unuei the expeiimental conuitions useu, to obtain the AuNPs.

The uetection limits achieveu iangeu fiom u.uu7 µmol L
-1
foi
uouecyl gallate anu u.uS µmol L
-1
foi ascoibic aciu. The compaiison of these
values with those iepoiteu in othei methous involving uiffeient uetection
systems |1Sj, shows that the uevelopeu methou notably impioves these
values. Foi instance, the uetection limits uesciibeu foi piopyl anu octyl
gallates aie u.29 anu 2.S µmol L
-1
, iespectively, while the values obtaineu in
the pioposeu methou aie u.u1 anu u.uu8 µmol
-1
, iespectively.

The methou has been successfully applieu to the analysis of seveial
fooustuffs achieving similai iesults that those obtaineu by liquiu
chiomatogiaphy anu, in the case of ascoibic aciu, by ieuox titiation.

Beteimination of polyphenolic content in beveiages using laccase, golu
nanopaiticles anu long wavelength fluoiimetiy

The uevelopment of methous to ueteimine total polyphenol content
in foous is an aiea of gieat inteiest. The most common methous aie the
official A0AC photometiic methou (Folin-Ciocalteu) |17j anu methous
baseu on ampeiometiic biosensois |18-19j. The pioposeu methou offeis
an innovative alteinative that is baseu in foui elements that uiffeientiate it
fiom the methous pieviously uesciibeu:

1) The use of long wavelength fluoiimetiy as uetection system,
which impioves the selectivity of the methou avoiuing
inteifeiences fiom backgiounu sample matiix.
Capitulo S
24u
2) The use of the enzyme laccase that offeis highei selectivity foi
the oxiuation of polyphenols, insteau of ieagents that act
against any ieuuctant piesent in the sample, such as the Folin-
Ciocalteu ieagent.
S) The methou is baseu on the tempoial inhibition causeu by
polyphenolic compounus on the oxiuation of the long
wavelength fluoiophoi inuocyanine gieen (λ
ex
764 nm, λ
em
8u6
nm), using a stoppeu-flow mouule to automate the mixing of the
ieactants anu the measuiement of the inuuction time, which is
piopoitional to the analyte concentiation.
4) The use of positively chaigeu AuNPs, which inteiact with the
enzyme anu ueciease its activity, allowing the impiovement of
the uetection limits obtaineu.

These elements have given iise to the uevelopment of a methou that
shows lowei uetection limits than those obtaineu in most of the methous
involving the use of ampeiometiic biosensois (Table 7).

Table 7. L0Bs compaiison between seveial methous

Analyte
Pioposeu
methou

|18j |19j |2uj

L0B
(µmol L
-1
)
L0B
(µmol L
-1
)
L0B
(µmol mL
-1
)
L0B
(µmol mL
-1
)
Catechol u.u1 1u -
-
Byuioquinone u.u1 - -
-
Byuioxyhyuioquinone u.uS - - -
uallic aciu u.u4 S8u u.41 17.u1
Pyiogallol u.u4 - - -


0ne limitation of the methou, as it occuis in othei methous foi total
polyphenol ueteimination, is that only a iough estimate can be achieveu
because the iesults aie iefeiieu to a polyphenol that is useu as a mouel, in
this case gallic aciu. Anothei ciitical aspect is the use of the enzyme in a
homogeneous meuium, unlike enzymatic biosensois in which the enzyme is
immobilizeu anu allows its ieusability. Bowevei, the amount of enzyme
useu in each measuiement is extiemely low uue to the high sensitivity of
the methou.
Biscussion of the iesults
241
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68, SS11-SS1S.
|6j Belajová, E.; Suhaj, N.; Foou Chem. 2uu4, 86, SS9-S4S.
|7j Caieii, N.; Elviii, L.; Nangia, A.; Nusci, N.; }. Chiomatogi. A, 2uuu,
881, 449-46u.
|8j Nata-Bilbao, N. L.; Anuiés-Lacueva. C.; }áuiegui, 0.; Lamuela-
Raventos, R.N.; Foou Chem. 2uu7, 1u1:1742-1747.
|9j }ustesen, 0.; Knuthsen, P.; Leth, T.; }. Chiomatogi. A, 1998, 799, 1u1-
11u.
|1uj Nouly, P.; uayuou, E. N.; Auffiay, A.; }. Chiomatogi. A, 1998, 8uu,
171-179.
|11j Abau-uaicia, B.; Beiiueta, L.A.; Lopez- Náiquez, B.N.; Ciespo-
Feiiei, I.; uallo, B.; vicente, F.; }. Chiomatogi. A, 2uu7, 11S4, 87-96.
|12j Nakiis, B. P.; Kallithiaka, S.; Namalos, A.; Talanta, 2uu6, 7u, 114S-
11S2.
|1Sj Beltián, N. B.; Buaite-Neimouu, N. A.; Bustos, N.A.; Salah, S. A.;
Loyola, E. A.; Peña-Neiia, A. I.; }alocha. }. W.; }. Foou Eng. 2uu6, 7S, 1-
1u.
|14j Bugo, P.; Piesti, N. L.; 0hman, N.; Fazzio, A.; Bugo, u.; Nonuello, L.; }
Sep. Sci. 2uuS, 28, 1149-11S6.
|1Sj Anuie , C.; Castanheiia, I.; Ciuz, }. N.; Paseiio, P.; Sanches-Silva, A.;
Tienus Foou Sci. Technol. 2u1u, 21, 229-246.
|16j Xiu-Qin, L; Chao, }.; Yan-Yan, S.; Nin-Li, Y.; Xiao-uang, C.; Foou Chem.
2uu9, 11S,692-7uu.
|17j Singleton, v. L.; Rossi, }. A.; Am., }.; Enol. viticult. 196S, 16, 144-1S8.
Capitulo S
242
|18j ElKaoutit, N.; Naianjo-Rouiiguez, I.; Temsamani, K. R.; Beinánuez-
Aitiga, N. P.; Belliuo-Nilla, B.; Biualgo-Biualgo ue Cisneios, }. L.;
Foou Chem. 2uu8, 11u, 1u19-1u24.
|19j Nonteieali, N. R.; Bella Seta, L.; vastaiella, W.; Pilloton, R.; }. Nol.
Cat. B: Enzymatic, 2u1u, 64, 189-194.
|2uj Bi Fuscoa, N.; Toitolinia, C.; Beiiua, B.; Nazzei, F.; Talanta, 2u1u, 81,
2SS-24u.






































CONCLUSIONES
CONCLUSIONS



























24S
CONCLUSIONES


En la Nemoiia piesentaua se han piopuesto uiveisas metouologias
analiticas, ciomatogiáficas y no ciomatogiáficas, paia la ueteiminacion ue
antioxiuantes natuiales y sintéticos en alimentos con el fin ue ampliai la
uisponibiliuau ue métouos aplicables al contiol ue la caliuau alimentaiia. A
continuacion se iesumen las piincipales conclusiones uel tiabajo iealizauo:

1. Se ha uesaiiollauo un métouo meuiante ciomatogiafia ue
liquiuos paia la ueteiminacion ue uieciocho compuestos fenolicos
en vinos utilizanuo meuiuas ue absoibancia a cuatio longituues
ue onua, y ue luminiscencia sensibilizaua ue teibio. Se ha
uemostiauo la utiliuau ue este último sistema ue ueteccion,
meuiante ueiivatizacion postcolumna ue los analitos, paia
mejoiai la selectiviuau ue estas ueteiminaciones.

2. Se ha uesciito un pioceuimiento paia el análisis clasificatoiio ue
vinos utilizanuo su conteniuo en compuestos fenolicos, su
vaiieuau y su oiigen geogiáfico. Los uatos obteniuos meuiante la
sepaiacion ciomatogiáfica ue los analitos y su ueteccion
fotométiica y luminiscente se han sometiuo a uiveisos
algoiitmos, aplicanuo métouos supeivisauos y no supeivisauos
ue pautas, consiguienuo la coiiecta uisciiminacion entie los
uistintos giupos estableciuos.

S. Se ha piopuesto un métouo paia la ueteiminacion ue flavonoiues
en zumos ue naianja basauo en su sepaiacion meuiante
ciomatogiafia ue liquiuos, ueiivatizacion postcolumna ue los
analitos con aluminio(III) y teibio(III) y ueteccion ue la
luminiscencia sensibilizaua. Se uemuestia poi piimeia vez la
utiliuau uel uso conjunto ue uos iones metálicos como
ueiivatizantes paia mejoiai los limites ue ueteccion pieviamente
obteniuos paia la cuantificacion ue estos analitos meuiante otios
sistemas ue ueteccion, tales como fotometiia, coulombimetiia o
espectiometiia ue masas.

4. Se ha uesciito poi piimeia vez el uso conjunto uel fluoiofoio ue
laiga longituu ue onua violeta ue ciesilo y ceiio(Iv) en meuio
micelai como sistema ue ueiivatizacion postcolumna en
ciomatogiafia ue liquiuos paia la ueteiminacion ue flavonoiues
en zumos ue naianja. La metouologia uesaiiollaua piesenta las
caiacteiisticas siguientes: 1) evita posibles inteifeiencias ue la

246
matiiz ue la muestia, uebiuo a la laiga longituu ue onua utilizaua
paia iealizai las meuiuas, 2) mejoia los limites ue ueteccion
obteniuos meuiante ueteccion fotométiica, y S) peimite el
análisis uiiecto ue las muestias uebiuo al elevauo factoi ue
uilucion que pueue utilizaise.

S. Se ha estuuiauo la capaciuau ue uiveisos compuestos
antioxiuantes utilizauos como auitivos natuiales o sintéticos en
alimentos paia foimai nanopaiticulas ue oio. El estuuio iealizauo
se ha aplicauo a la ueteiminacion cinética ue estos compuestos,
utilizanuo un sistema ue flujo ueteniuo, meuiante monitoiizacion
ue la iauiacion uispeisaua piouuciua poi las nanopaiticulas
foimauas. El métouo piopuesto evita las etapas ue incubacion
iequeiiuas en métouos ue equilibiio y peimite obtenei
iesultauos analiticos en pocos segunuos. El métouo se ha aplicauo
al análisis ue uiveisos alimentos y los iesultauos se han valiuauo
utilizanuo uos métouos ue iefeiencia.

6. Se ha investigauo la utiliuau analitica ue la fluoiimetiia ue laiga
longituu ue onua paia la ueteiminacion ue polifenoles en bebiuas
utilizanuo la inhibicion tempoial piouuciua poi estos compuestos
en la oxiuacion uel fluoiofoio veiue ue inuocianina, en piesencia
ue la enzima laccasa y ue nanopaiticulas ue oio. Se ha
uemostiauo que la inteiaccion electiostática entie la enzima y las
nanopaiticulas mejoia los limites ue ueteccion obteniuos,
consiguienuo valoies infeiioies a los uesciitos en la mayoiia ue
los biosensoies ampeiométiicos pieviamente piopuestos paia la
ueteiminacion ue estos compuestos.
















247
CONCLUSIONS


Seveial analytical chiomatogiaphic anu non chiomatogiaphic
methouologies foi the ueteimination of natuial anu synthetic antioxiuants
in foous have been pioposeu in this stuuy. The puipose has been to expanu
the availability of methous foi the contiol of foou quality. The main
conclusions of the ieseaich peifoimeu aie summaiizeu below:


1. A liquiu chiomatogiaphic methou with online photometiic anu
luminescent uetection foi the ueteimination of eighteen phenolic
compounus in wines has been uevelopeu. Photometiic uetection
has been peifoimeu at foui uiffeient wavelengths anu the
luminescent uetection has been achieveu using teibium(III) as a
postcolumn ueiivatization ieagent. The usefulness of the last
uetection system to impiove the selectivity of these
ueteiminations has been uemonstiateu.

2. A methou foi wine classification baseu on the phenolic compounu
content, wine vaiiety anu geogiaphical aiea has been uesciibeu.
The uata obtaineu fiom the chiomatogiaphic sepaiation of the
analytes anu theii photometiic anu fluoiimetiic uetection have
been subjecteu to uiffeient algoiithms, applying supeiviseu anu
un-supeiviseu pattein iecognition methous, achieving the coiiect
uisciimination between the uiffeient gioups establisheu.

S. A methou foi the ueteimination of flavonoius in oiange juices,
involving liquiu chiomatogiaphic sepaiation, post-column
ueiivatization of the analytes with teibium(III) anu
aluminium(III) anu sensitizeu luminescence uetection, has been
uesciibeu. The usefulness of two metal ions as ueiivatizing agents
to impiove the uetection limits achieveu by otheis uetection
systems, such as photometiy, coulombimetiy anu mass
spectiometiy, is shown foi the fiist time.

4. A new methou foi the ueteimination of flavonoius in oiange
juices has been uesciibeu, which is baseu on the simultaneous
use of the long wavelength fluoiophoi ciesyl violet anu
ceiium(Iv) in a micellai meuium as a post-column ueiivatizing
system in liquiu chiomatogiaphy. The main featuies of the

248
methou aie the following: 1) the use of long wavelength
measuiements to obtain analytical signals avoius the inteifeience
of potential signals fiom the sample matiix, 2) the uetection
limits achieveu in this methou aie bettei than those obtaineu
using photometiic uetection, anu S) the methou allows the uiiect
analysis of the samples owing to the high uilution factois that can
be useu.

S. The capability of seveial antioxiuant compounus, useu as natuial
oi synthetic auuitives in foou, to ieuuce golu(III) to golu
nanopaiticles has been stuuieu. This stuuy has been applieu to
the kinetic ueteimination of these compounus using stoppeu-
flow mixing technique anu monitoiing the foimation of golu
nanopaiticles using iesonance light scatteiing as the uetection
system. The pioposeu methou avoius incubation steps in contiast
to equilibiium methous anu the analytical iesults can be achieveu
in few seconus. The methou has been applieu to the analysis of
seveial foou samples anu the iesults obtaineu weie valiuateu
using two iefeience methous.

6. The analytical utility of long wavelength fluoiimetiy foi the
ueteimination of poliphenols compounus in beveiages has been
stuuieu. A methou baseu on the tempoial inhibition causeu by
these compounus on the oxiuation of the fluoiophoi inuocyanine
gieen in the piesence of the enzyme laccase anu positive chaigeu
golu nanopaiticles has been uevelopeu. It has been shown that
the electiostatic inteiaction between the enzyme anu the
nanopaiticles impioves the uetection limits obtaineu, achieving
values that aie lowei than those obtaineu in most of the methous
baseu on ampeiometiic biosensois pioposeu foi the
ueteimination of these compounus.

ANLkC
ÞkCDUCCICN CILN1IIICA

2S1
Producciún científica derivada de esta Tesis Doctoral

Publicaciones:

1. Analytical innovations in the uetection of phenolics in wines.
Russo, P.; Anuieu-Navaiio, A.; Aguilai-Caballos, N. P.; Feinánuez-
Romeio, }. N.; uomez-Bens, A.
}ouinal of Agiicultuial anu Foou Chemistiy. 2uu8, S6, 18S8-186S.

2. 0sefulness of teibium-sensitiseu luminescence uetection foi the
chemometiic classification of wines by theii content in phenolic
compounus.
Anuieu-Navaiio, A; Russo, P.; Aguilai-Caballos, N. P.; Feinánuez-
Romeio, }. N.; uomez-Bens, A.
Foou Chemistiy, 2u11, 124, 17SS-17S9.

S. Luminescent ueteimination of flavonoius in oiange juices by LC
with post-column ueiivatization with aluminum anu teibium.
Anuieu-Navaiio, A.; Feinánuez- Romeio, }. N.; uomez-Bens, A.
}ouinal of Sepaiation Science, 2u1u, SS, Su9-S1S.

4. Long-Wavelength Fluoiescence Betection of Flavonoius in 0iange
}uices by LC.
Anuieu-Navaiio, A.; Feinánuez- Romeio, }. N.; uomez-Bens, A.
Chiomatogiaphia, 2u1u, 72, 111S-112u.

S. Beteimination of antioxiuant auuitives in fooustuffs by uiiect
measuiement of golu nanopaiticle foimation using iesonance light
scatteiing uetection.
Anuieu-Navaiio, A.; Feinánuez- Romeio, }. N.; uomez-Bens, A.
Analytica Chimica Acta 69S (2u11) 11-17.

6. Beteimination of polyphenolic content in beveiages using laccase,
golu nanopaiticles anu long wavelength fluoiimetiy.
Anuieu-Navaiio, A.; Feinánuez- Romeio, }. N.; uomez-Bens, A.
Sent to Analytical Chimica Acta on 0ctobei 2u11.


Analytical Innovations in the Detection of Phenolics
in Wines
PIETRO RUSSO, ÁLVARO ANDREU-NAVARRO, MARÍA-PAZ AGUILAR-CABALLOS,
JUAN-MANUEL FERNÁNDEZ-ROMERO, AND AGUSTINA GÓMEZ-HENS*
Department of Analytical Chemistry, “Marie Curie Annex” building, Campus of Rabanales, University
of Córdoba, E-14071 Córdoba, Spain
A liquid chromatographic method with online photometric and luminescent detection for the
determination of 18 phenolic compounds in wines is reported. Photometric detection is performed at
four wavelengths, namely, 256, 280, 320, and 365 nm, using a diode array detection system. The
luminescent detection is achieved by means of a postcolumn derivatization reaction of 10 of these
compounds with terbium(III) in the presence of synergistic agents, such as ethylenediaminetetraacetic
acid (EDTA) and n-octyltriphosphine oxide (TOPO). A micellar medium provided by the surfactants
sodium dodecyl sulfate and Triton X-100 was used for the determination of the luminescent chelates
at λ
ex
317 and λ
em
545 nm. The long wavelength emission of lanthanide chelates can minimize
interferences frombackground sample matrix, which usually emit at shorter wavelengths. The analytical
features of the photometric and fluorometric methods, such as dynamic ranges of the calibration
graphs, detection limits, and precision data, have been obtained. The practical usefulness of the
developed methods is demonstrated by the analysis of Spanish and Italian wine samples (red, rosé,
oloroso, and white), which were diluted and directly injected into the chromatographic system. The
accuracy of both methods was checked by assaying a recovery study, which was performed at three
different analyte levels for each type of sample.
KEYWORDS: Phenolic compounds; postcolumn derivatization; terbium-sensitized luminescence; wine
samples
INTRODUCTION
Phenolics are a wide group of compounds constituted by
phenolic aldehydes, hydroxybenzoic and hydroxycinnamic acids,
catechins, flavonols, and stilbenes, in their monomeric form or
conjugated to some species, such as tartaric acid in the case of
cinnamic acids (1), among others. These compounds are present
in wines because they are secondary metabolites of plants. The
composition of phenolics and their concentration depend on
grape variety, geographical origin, soil type, collection system,
and grape processing. These compounds are responsible of the
sensory properties of the wines, and, also, they are anticarci-
nogenic and have an anti-inflammatory action when they are
regularly ingested. A particular example of the importance of
monitoring phenolic concentration to control the quality of wines
is the presence of aromatic aldehydes, formed by a group of
volatile compounds that are extracted from wood lignin during
the winemaking process. The presence of these aldehydes in
wines is an indicator of fermentation and aging in oak barrels,
their absence being indicative of counterfeit aged wines. Vanillin
and syringaldehyde are the most abundant aromatic aldehydes
in wines.
The occurrence of phenolics has been extensively studied by
liquid chromatographic methods (1–18). In most of them,
conventional reversed-phase columns, constituted by packed
microparticulate bonded silica, have been used (1–5, 7–15, 18),
which generally feature separations of 14–25 compounds in
almost 1 h or 32 phenolics in 90 min, which makes routine
analysis of these compounds very tedious. The use of other
materials such as mesoporous silica has given rise to monolithic
columns, which operate at higher flow rates with lower back-
pressures than conventional columns (16, 17). Thus, they allow
the analysis of samples using direct injection or low sample
dilutions, because the cleaning and regeneration of the column
can be done more quickly than in the conventional ones due to
the high flow rates afforded. Monolithic columns have been used
for the determination of 15 phenolics in red and white wine
samples (16) with separation times around 30 min and, also,
for the direct analysis of cider samples (19).
Diode array detection (1, 3–17) has been extensively used
for the development of liquid chromatography methods, whereas
fluorometric (8, 12) and mass spectrometry (2) detection systems
have been used to a lesser extent. Most fluorometric methods
proposed are based on measurements of the intrinsic fluores-
cence of some phenolics. Although these methods feature
generally lower detection limits than photometric methods, the
* Author to whom correspondence should be addressed (telephone
+ 34-957218645; fax +34-957-218644; e-mail qa1gohea@uco.es).
1858 J. Agric. Food Chem. 2008, 56, 1858–1865
10.1021/jf073206t CCC: $40.75 © 2008 American Chemical Society
Published on Web 02/22/2008
Analytical Methods
Usefulness of terbium-sensitised luminescence detection for the chemometric
classification of wines by their content in phenolic compounds
A. Andreu-Navarro, P. Russo, M.P. Aguilar-Caballos, J.M. Fernández-Romero, A. Gómez-Hens

Department of Analytical Chemistry, ‘‘Marie Curie Annex” Building, Campus of Rabanales, University of Córdoba, E-14071 Córdoba, Spain
a r t i c l e i n f o
Article history:
Received 4 May 2009
Received in revised form 2 August 2010
Accepted 9 August 2010
Keywords:
Phenolic compounds
LC-photometry and fluorimetry
Terbium(III)
Factor Analysis (FA)
Linear discriminant analysis (LDA)
Wine samples
a b s t r a c t
A method for wine classification based on the phenolic compound content, wine variety and geographical
area is described. The method involves the use of the results obtained from the analysis of fifteen samples
of Italian and Spanish wines from different geographical origins [Sicilia (Italy) and Córdoba (Spain)] using
liquid chromatography (LC) with photometric and fluorimetric detection, in which eighteen phenolics
were determined: gallic acid, protocatechuic acid, p-hydroxybenzoic acid, salicylic acid, vanillic acid, caf-
feic acid, syringic acid, catechin, vanillin, p-coumaric acid, syringaldehyde, epicatechin, ferulic acid, rutin,
trans- and cis-resveratrol, quercetin and kaempferol. Photometric measurements were performed select-
ing four wavelengths (256, 280, 320 and 365 nm), using a diode-array detection system. The fluorimetric
detection was achieved by measuring the sensitised luminescence provided by the chelates formed
between each analyte and terbium (III). All samples were commercial wines bought in local markets
and analysed immediately after they were opened. The pattern data matrix was constructed by the con-
centration of each analyte present in wine, which was determined by the most adequate method, namely
LC-photometric or LC-fluorimetric method. This data matrix was subjected to different algorithms in
order to classify and characterise the wine samples adequately. Supervised (LDA) and un-supervised
(FA) pattern recognition methods were used. The wine pattern generation with LC separation and dual
detection approach to determine eighteen phenolic compounds and the chemometric treatment provide
an appropriate way with recognition and prediction rates. The values obtained for these rates were 100%
when fluorimetric detection was used. These results can be considered satisfactory, which proves the
usefulness of the selected variables.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
In recent years, there has been an increasing interest in the
development of chemometric applications in order to establish
the variety, geographical origin, manipulation and other techno-
logical features, which define the taste of foods and beverages from
agricultural origin (Ariyama & Yasui, 2006; Marcos, Fischer, Rea, &
Hill, 1998; Sanz, Pérez, Herrera, Sanz, & Juan, 1995; Smith, 2005).
The reasons for this interest are: (1) laws enforce labelling of the
geographical origin of the foodstuff and beverages in many coun-
tries due to demands of more information from consumers and
improvement of their domestic production; (2) producers have be-
gun to advertise their brands of high-quality products by including
geographical origin and other features in the label for economic
reasons; (3) prevention of frauds and adulteration on the label,
production and commercialisation. Thus, the development of
methods giving acceptable information is highly desirable for con-
sumers, producers and administrative authorities.
Wine industry and the market sector are particular examples in
which the development of sophisticated chemometric techniques
are essential for the improvement of the analytical information
about wine composition and for assessing wine authenticity
(Arvanitoyannis, Katsota, Psarra, Soufleros, & Kallithraka, 1999;
Horwitz, 1995). Several chemometric procedures have been used
as the basis for discrimination of wines according to vinification
technology and classification according to region, type and variety.
Different pattern recognition techniques (PRT), such as Principal
Component Analysis (PCA) (Boselli, Giomo, Minardi, & Frega,
2008; Recamales, Sayago, González-Miret, & Hernanz, 2006), Linear
and Canonical Discriminant Analysis (LDA and CDA) (Hernández,
Estrella, Dueñas, Fernandez de Simón, & Cadahía, 2007; Makris,
Kallithraka, & Mamalos, 2006; Villiers et al., 2005), Probabilistic
Neural Network (PNN) (Díaz, Conde, Estévez, Pérez-Olivero, &
Pérez Trujillo, 2003), K-Nearest Neighbours (KNN) (Beltrán et al.,
2006), Cluster Analysis (CA) (Villiers et al., 2005), Multiregression
Analysis (MRA), Partial Least Squares (PLS) (Capron, Smeyers-
Verbeke, & Massart, 2007; Le Moigne et al., 2008), and CAIMAN
0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.08.014

Corresponding author. Tel.: +34 957218645; fax: +34 957218644.
E-mail address: qa1gohea@uco.es (A. Gómez-Hens).
Food Chemistry 124 (2011) 1753–1759
Contents lists available at ScienceDirect
Food Chemistry
j our nal homepage: www. el sevi er . com/ l ocat e/ f oodchem
Research Article
Luminescent determination of flavonoids in
orange juices by LC with post-column
derivatization with aluminum and terbium
A new post-column derivatization system is described and applied to the determination of
flavonoids in citric beverages after their separation by LC using a monolithic column. The
derivatization involves the formation of the chelates of the analytes with aluminum (III)
and terbium (III) in the presence of the surfactant SDS and the measurement of the
terbium sensitized luminescence at l
ex
360 and l
em
545 nm. Naringin, hesperidin,
quercetin, naringenin, and kaempferol have been chosen as analyte models. The large
Stokes shift and the relatively long wavelength emission of terbium(III) can minimize
interferences from background sample matrix, which usually emit at shorter wavelengths.
Calibration graphs were constructed in the intervals 6.0–1700 ng/mL naringin,
9.8–1700 ng/mL hesperidin, 2.1–2000 ng/mL quercetin, 5.2–1500 ng/mL naringenin and
2.5–2000 ng/mL kaempferol, with regression coefficients higher than 0.9935 in all
instances. The precision of the method, expressed as RSD%, was established at two
concentration levels, with values of 1.3 and 4.7%, which corresponded to the minimal and
maximal error zones of the calibration graphs. The practical usefulness of the method is
demonstrated by the analysis of orange juices, which were diluted and directly injected
into the chromatographic system, obtaining recoveries between 86.9 and 108.2%.
Keywords: Aluminum and terbium complexes / Flavonoid compounds / Orange
juices / Post-column derivatization
DOI 10.1002/jssc.200900696
1 Introduction
Flavonoids are a group of polyphenolic compounds widely
distributed in vegetables and fruits [1]. The determination of
these compounds is of great interest owing to their multiple
biological effects, including antioxidant activity, antitumor,
antimutagenic, antibacterial and angioprotective properties
[2, 3]. They also contribute to different plant properties such
as colour, flavour, fragrance, nutrition, stability and
therapeutic properties. Flavonoids such as 2-phenyl-benzo-
a-pyrones are classified according to the multitude of
substitution patterns in the two benzene rings of their
basic structure. Variation in their heterocyclic rings gives
rise to flavonols, flavones, catechins, flavanones, anthocya-
nidins and isoflavones [4]. The flavonoid content in orange
juices has special interest as it contributes to the quality of
these samples [5].
The main separation technique applied to the determi-
nation of flavonoids in orange juices is LC [6–11], using
reversed-phase columns, although gas chromatography [12],
involving the formation of trimethylsilyl derivatives, has
been also described for this purpose. The usefulness of a
monolithic column, which can operate at higher flow rates
with lower backpressures than conventional columns, has
been previously described for the LC separation of some
flavonoids in wine samples [13]. However, to the best of our
knowledge, this type of column has not been used up to date
for the determination of these compounds in orange juices.
Photometric [6–10], coulometric [10] and MS [6, 11]
detection systems have been reported in the LC methods
described for the analysis of these samples. Some flavo-
noids, such as quercetin and kaempferol, have been fluor-
imetrically determined in body fluids using aluminum(III)
as post-column derivatization reagent to obtain fluorescent
chelates [14]. Another post-column derivatization system
previously described for the determination of these
compounds in wine samples involves the formation of the
corresponding terbium(III) chelates and the measurement
of the sensitized luminescence [13]. This phenomenon is an
intermolecular energy transfer process, in which the ligand
acts as donor and terbium(III) acts as acceptor and emits
luminescence, that allows very sensitive and selective
determinations, although its use as detection system in LC
has been relatively limited [15].
This article reports for the first time the use of a
monolithic column for the direct separation of flavonoids in
orange juice samples and a new post-column derivatization
Alvaro Andreu-Navarro
Juan Manuel Ferna´ ndez-
Romero
Agustina Go´ mez-Hens
Department of Analytical
Chemistry, Faculty of Sciences,
University of Co´ rdoba, Co´ rdoba,
Spain
Received October 23, 2009
Revised December 18, 2009
Accepted December 21, 2009
Correspondence: Professor A. Go´ mez-Hens, Department
of Analytical Chemistry, Faculty of Sciences, University of
Co´ rdoba, ‘‘Marie Curie Annex’’ Building, Campus of Rabanales,
E-14071 Co´ rdoba, Spain
E-mail: qa1gohea@uco.es
Fax:134-957-218644
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
J. Sep. Sci. 2010, 33, 509–515 509
Long-Wavelength Fluorescence Detection
of Flavonoids in Orange Juices by LC
Alvaro Andreu-Navarro, Juan Manuel Ferna´ndez-Romero, Agustina Go´mez-Hens
&
Department of Analytical Chemistry, Faculty of Sciences, University of Co´rdoba, ‘‘Marie Curie Annex’’ Building,
Campus of Rabanales, 14071 Co´rdoba, Spain; E-Mail: qa1gohea@uco.es
Received: 16 April 2010 / Revised: 7 September 2010/ Accepted: 7 September 2010
Abstract
A new post-column liquid chromatographic reaction system for the determination of flavo-
noids in orange juices is based on the use of the long wavelength fluorophor cresyl violet and
cerium(IV) in a cetyl trimethylammonium bromide micellar medium. Two flavone aglycones
(quercetin and kaempferol), a flavonone aglycone (naringenin), one flavone-O-glycoside
(rutin) and two flavanone-O-glycosides (hesperidin and naringin) have been used as analyte
models. The reaction process involves the interaction between the analyte, cerium(IV) and
cresyl violet giving rise to a decrease in the fluorescence, measured at k
ex
585, k
em
625 nm,
which is proportional to the analyte concentration. Dynamic ranges of the calibration graphs
and detection limits, obtained with standard solutions of the analytes are (ng mL
-1
): quer-
cetin (12.2–4,000, 3.7), kaempferol (3.5–1,000, 1.0), naringenin (6.7–1,000, 2.0), rutin
(5.0–800, 1.5), hesperidin (10.1–1,000, 3.0), and naringin (17.8–800, 1.8). The deter-
mination coefficients were higher than 0.993 in all instances. The precision of the method,
expressed as RSD%, was established at two concentration levels, with values ranging
between 2.8 and 6.2%. The practical usefulness of the developed method is demonstrated by
the analysis of natural and commercial orange juices, which were filtered, diluted and
directly injected into the chromatographic system, with apparent recoveries between 86.9
and 107.0%.
Keywords
Column liquid chromatography
Long wavelength fluorescence detection
Flavonoid compounds
Orange juice samples
Introduction
The use of long-wavelength fluorophores
(LWF) as analytical reagents is an
attractive option to improve the selectiv-
ity of fluorimetric analysis and avoid or
minimize the sample treatment step of the
analytical process. These dyes allowing
measurements are obtained in a region of
the electromagnetic spectrum (>600 nm)
in which the potential absorption or
emission associated to sample matrix is
minimized. Also, Raman interference is
greatly diminished and the probability of
non-radiative quenching processes is
decreased due to the usually short fluo-
rescence lifetime of these fluorophores.
LWFs have been described as enzymatic
substrates, immunoassay labels, sensing
systems and derivatizing reagents in
liquid chromatography (LC) and capil-
lary electrophoresis [1]. Although the lack
of sufficient reactive groups for targeting
of analytes is a limitation of these fluo-
rophores, their analytical usefulness has
been expanded using electrostatic and
redox interactions [2–6].
This article describes for the first time
the use of the oxazine LWF cresyl violet
(CV) in a post-column LC reaction
system for the indirect determination of
DOI: 10.1365/s10337-010-1774-8
Ó 2010 Vieweg+Teubner Verlag | Springer Fachmedien Wiesbaden GmbH
Original
Author's personal copy
Analytica Chimica Acta 695 (2011) 11–17
Contents lists available at ScienceDirect
Analytica Chimica Acta
j our nal homepage: www. el sevi er . com/ l ocat e/ aca
Determination of antioxidant additives in foodstuffs by direct measurement of
gold nanoparticle formation using resonance light scattering detection
A. Andreu-Navarro, J.M. Fernández-Romero, A. Gómez-Hens

Department of Analytical Chemistry, Faculty of Sciences, University of Córdoba, “Marie Curie Annex” Building, Campus of Rabanales, E-14071 Córdoba, Spain
a r t i c l e i n f o
Article history:
Received 21 January 2011
Received in revised form 14 March 2011
Accepted 24 March 2011
Available online 1 April 2011
Keywords:
Antioxidant additives
Gold nanoparticles
Stopped-flow mixing technique
Resonance light scattering detection
Foodstuff samples
a b s t r a c t
The capability of antioxidant compounds to reduce gold(III) to gold nanoparticles has been kinetically
studied in the presence of cetyltrimethylammonium bromide using stopped-flow mixing technique and
resonance light scattering as detection system. This study has given rise to a simple and rapid method for
the determination of several synthetic and natural antioxidants used as additives in foodstuff samples.
The formation of AuNPs was monitored by measuring the initial reaction-rate of the system in about
5s, using an integration time of 0.1s. Dynamic ranges of the calibration graphs and detection limits,
obtained with standard solutions of the analytes, were (␮mol L
−1
): gallic acid (0.04–0.59, 0.01), propyl
gallate (0.04–1.41, 0.01), octyl gallate (0.03–0.35, 0.08), dodecyl gallate (0.02–0.30, 0.007), butylated
hydroxyanisol (0.07–0.39, 0.009), butylated hydroxytoluene (0.04–0.32, 0.01), ascorbic acid (0.11–1.72,
0.03) and sodium citrate (0.07–1.29, 0.02). The regression coefficients were higher than 0.994 in all
instances. The precision of the method, expressed as RSD%, was established at two concentration levels
of each analyte, with values ranging between 0.6 and 4.8%. The practical usefulness of the developed
method was demonstrated by the determination of several antioxidant additives in foodstuff samples,
which were extracted, appropriately diluted and assayed, obtaining recoveries between 95.4 and 99.5%.
The results obtained were validated using two reference methods.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Natural and synthetic antioxidants are a group of compounds
used as additives to prevent or retard oxidation reactions in food
products. There is a trend to limit the use of synthetic antioxidants,
owingtotheir potential toxic effects, but theyare still foundina rel-
ativelywiderangeof foodstuffs, althoughtheir useis subject tovery
strict safety regulations [1–6]. Phenolic compounds such as propyl
(PG), octyl (OG) and dodecyl (DG) esters of gallic acid (GA), buty-
lated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT)
are the main synthetic antioxidants still authorized, although they
are being replaced by natural antioxidants, such as ascorbic (AA)
and citric (CA) acids. CA is also used as a food additive for other
purposes, such as acidifier and flavouring agent.
The determination of synthetic antioxidants in food samples
is mainly carried out by using reverse-phase liquid chromatogra-
phy (LC) with photometry or mass spectrometry (MS) as detection
systems [7,8]. Methods based on gas chromatography (GC), with

Corresponding author. Tel.: +34 957 218645; fax: +34 957 218644.
E-mail address: qa1gohea@uco.es (A. Gómez-Hens).
flame ionization detector (FID) or MS, and capillary electrophore-
sis (CE) with photometric or electrochemical detection have been
also described [7]. The limits of detection (LODs) reported for most
of these methods are at the level of ␮mol L
−1
. These methods are
very useful for the identification of the analytes, but they are time-
consuming for screening purposes.
The use of gold nanoparticles (AuNPs) as analytical reagents
has allowed the development of very sensitive methods based on
their special optical and electrochemical properties. For instance,
they have been used as labels in immunoassays and hybridiza-
tion assays and as nanoscaffolds to develop chemical sensors [9].
These methods require the previous synthesis of the NPs, using
generally tetrachloroauric acid and a reducing reagent, usually cit-
rate [10,11]. Other reagents such as cysteine [12], ascorbic acid
[10,13,14], sodiumborohydride [15,16] and gallic acid [17,18] have
alsoshowntheir usefulness for this purpose. The experimental con-
ditions chosen to obtain AuNPs are critical factors that affect the
size, shape and potential aggregation of the NPs and, consequently,
their properties. Some of these methods [14,15] involve the use of
cetyltrimethylammonium bromide (CTAB) to favour the synthesis
of the NPs. The formation of ion pairs between AuCl
4

and cationic
surfactants prior to the formation of AuNPs was described [19,20].
0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2011.03.047
1
Determination of polyphenolic content in beverages using
laccase, gold nanoparticles and long wavelength fluorimetry

A. Andreu-Navarro, J.M. Fernández-Romero, A. Gómez-Hens
(*)


Department of Analytical Chemistry, Faculty of Sciences, University of Córdoba, “Marie
Curie Annex” Building. Campus of Rabanales, E-14071 Córdoba, Spain



ABSTRACT

An enzymatic fluorimetric method for the determination of polyphenol compounds
in beverages is described, which is based on the temporal inhibition caused by these
compounds on the oxidation of the long wavelength fluorophor indocyanine green (
ex
764
nm,
em
806 nm), in the presence of the enzyme laccase and positive charged gold
nanoparticles (AuNPs). The oxidation of the dye gives rise to a fast decrease in its
fluorescence, but it is delayed by the polyphenol, obtaining a time period directly
proportional to its concentration, which has been used as the analytical parameter. The
behaviour of several benzenediols and benzenetriols in the system and the modification of
the activity of the enzyme by its interaction with AuNPs have been studied.
The system has been optimized using gallic acid as a polyphenol model, but the
dynamic ranges of the calibration graphs and the detection limits for several of the
polyphenols assayed were obtained (mol L
-1
): gallic acid (0.13-5, 0.04), catechol (0.08-5,
0.01), hydroquinone (0.05-2, 0.01), hydroxyhydroquinone (0.09-5, 0.03), pyrogallol (0.17-
5, 0.04). Most of the values of the regression coefficients were 0.999 and the precision of
the method, expressed as RSD% and checked at two concentration levels of each analyte,
ranged between 1.8 and 5.6%. The method has been applied to the determination of
polyphenol content in several foodstuff samples and the results compared with those
obtained with the standard Folin-Ciocalteu method.


KEYWORDS: Polyphenols; laccase; gold nanoparticles; stopped-flow mixing
technique; long wavelength fluorimetric detection; beverage samples

* Author to whom correspondence should be addressed (telephone +34 957 218645; fax
+34 957 218644. E-mail address: qa1gohea@uco.es )
2S8
Comunicaciones a congiesos:

1. 12
as
}oinauas ue Análisis Instiumental.
Baicelona, 21-2S 0ctubie ue 2uu8
Chiomatogiaphic ueteimination of nine flavonoius using
fluoiescence iesonance eneigy tiansfei as post-column
ueiivatization system.
Alvaio Anuieu-Navaiio, }uan N. Feinánuez-Romeio, Agustina
uomez-Bens.

2. II Encuentio sobie Nanociencia y Nanotecnologia ue investigauoies
y tecnologos ue la univeisiuau ue Coiuoba (NAN00C0).
Coiuoba, 14 ue Eneio ue 2u1u.
Kinetic stuuy of the foimation of coloiual golu nanopaiticles by
gallic aciu using stoppeu-flow mixing uevice anu light scatteiing
uetection.
Alvaio Anuieu-Navaiio, }uan N. Feinánuez-Romeio, Agustina
uomez-Bens.

S. XII Reunion uel uiupo Regional Anualuz ue la Socieuau Española ue
Quimica Analitica (uRASECA).
Coiuoba, 1u y 11 ue }unio ue 2u1u.
Beteiminacion uiiecta ue flavonoiues en zumos ue naianja
meuiante ciomatogiafia ue liquiuos y ueteccion fluoiescente a laiga
longituu ue onua.
Alvaio Anuieu-Navaiio, }uan N. Feinánuez-Romeio, Agustina
uomez-Bens.

4. III Encuentio sobie Nanociencia y Nanotecnologia ue
investigauoies y tecnologos ue la univeisiuau ue Coiuoba
(NAN00C0).
Coiuoba, 1u y 11 ue Febieio ue 2u11.
Beteimination of antioxiuant auuitives in fooustuffs by uiiect
measuiement of golu nanopaiticle foimation using iesonance light
scatteiing uetection.
Alvaio Anuieu-Navaiio, }uan N. Feinánuez-Romeio, Agustina
uomez-Bens.






2S9
S. 1S
as
}oinauas ue Análisis Instiumental.
Baicelona, 14-18 Noviembie ue 2u11
Fluoiimetiic ueteimination of polyphenols using a long-
wavelenght-fluoiophoi anu laccase immobiliseu on golu
nanopaiticles.
Alvaio Anuieu-Navaiio, }uan N. Feinánuez-Romeio, Agustina
uomez-Bens














12
as
1ORNADAS DE ANÁLISIS
INSTRUMENTAL




LIBRO DE RESUMENES










Barcelona
21-23 de Octubre de 2008


Poster 12
as
Jornadas de Analisis Instrumental (JAI)

144 Calidad y Seguridad Alimentaria

PO-CSA-53

CHROMATOGRAPHIC DETERMINATION OF NINE FLAVONOIDS USING
FLUORESCENCE RESONANCE ENERGY TRANSFER AS POST-COLUMN
DERIVATIZATION SYSTEM

Álvaro Andreu-Navarro, 1uan M. Fernández-Romero and Agustina Gómez-Hens.

Department of Analvtical Chemistrv, Facultv of Sciences, Universitv of Cordoba, 'Marie Curie
Annex` Building. Campus of Rabanales, E-14071 Cordoba, Spain. Email. q02annaa¸uco.es phone.
34-957-218645, fax. 34-957-218644

A liquid chromatographic method with on-line luminescent detection Ior the determination oI nine
antioxidative phenolic (galic, caIIeic and p-coumaric acids) and Ilavonoid (naringenin, naringuin,
hesperidin, quercetin, rutin and kaempIerol) compounds is reported. The post-column derivatization
system is based on the Iormation oI the Iluorescent chelates oI these compounds with aluminium(III),
which act as energy donors, and the energy transIer process Irom these chelates to terbium(III), which
acts as an energy acceptor. A micellar medium provided by the surIactant sodium dodecylsulIate was
used Ior the luminescence detection at
ex
412,
em
545 nm. The long wavelength emission minimizes
potential interIerences Irom background sample matrix, which usually emit at shorter wavelengths.
The analytical Ieatures oI the Iluorimetric methods, such as dynamic ranges oI the calibration graphs,
detection limits and precision data have been obtained. The practical useIulness oI the developed
method is demonstrated by the analysis oI citric juices.


Córdoba, 14 de enero de 2010

II ENCUENTRO SOBRE
NANOCIENCIA Y NANOTECNOLOGÍA
DE INVESTIGADORES Y TECNÓLOGOS
DE LA UNIVERSIDAD DE CÓRDOBA

Universidad de
Córdoba
Instituto Andaluz
de Química Fina y Nanoquímica
Consejería de Innovación,
Ciencia y Empresa


KINETIC STUDY OF THE FORMATION OF COLLOIDAL GOLD
NANOPARTICLES BY GALLIC ACID USING STOPPED-FLOW MIXING
DEVICE AND LIGHT SCATTERING DETECTION


A. Andréu-Navarro, 1.M. Fernández-Romero and A. Gómez-Hens

Department of Analvtical Chemistrv, Marie Curie Annex Building, Campus of
Rabanales, Universitv of Cordoba, E-14071-Cordoba, Spain. qa1gohea¸uco.es,
http.//www.uco.es/investiga/grupos/FQM-303


The capability oI gallic acid to reduce gold (III) chloride trihydrate to colloidal gold
nanoparticles and the aggregation process in the presence oI the cationic surIactant
cetyltrimethyl ammonium bromide (CTAB) have been kinetically studied using a
stopped-Ilow mixing device and light scattering as detection system. This study has
given rise to a simple and rapid method Ior the determination oI gallic acid in natural
samples and can be extended to other antioxidant compounds. The aggregation process
has been monitored by measuring the initial reaction-rate (v
0
) and the light scattering
signal at a preIixed-time (IF
40
), using stopped-Ilow mixing technique, which makes
the method applicable to automate routine analysis. Each measurement was obtained in
about 60 s, using an integration time oI 0.1 s. The dynamic range oI the calibration
graph obtained Ior gallic acid, using the initial rate method, was 0.06 0.6 µM, and the
detection limit was 0.01 µM. The precision oI the method, expressed as relative
standard deviation, ranged between 0.9 and 4.8 °.

P-16
·
·
,
,
.

.

^··.. r ¸ rr · ¸...
REUNIÓN DEL GRUPO REGIONAL ANDALUZ DE LA
SOCIEDAD ESPAÑOLA DE QUÍMICA ANALÍTICA

Sesión de Carteles
92
SC-36

Determinación directa de flavonoides en zumos de naranja
mediante cromatografia de liquidos y detección fluorescente a
larga longitud de onda

Al·aro Andreu-Na·arro, Juan Manuel lernandez-Romero, Agustina Gómez-lens

Departmento de Química Analítica, Facultad de Ciencias. Universidad de Córdoba. Edificio “Anexo Marie Curie”
Cav¡v. ae Rabavate., í·110¨1 Córaoba, í.¡ava
1etefovo: ²1··:¨21ºó1:, ía·: ²1··:¨21ºó11, evait: q02avvaa¸vco.e.
btt¡:,,rrr.vco.e.,ivre.tiga,grv¡o.,íQM·²0²

Se describe un nue·o método para la determinación de íla·onoides en zumos de naranja mediante
separación cromatograíica y deri·atización post-columna con detección íluorimétrica a larga longitud
de onda. Ll sistema propuesto se basa en el uso de ·ioleta de cresilo y cerio,IV, en un medio micelar
de bromuro de cetiltrimetil amonio. Los íla·onoides utilizados como analitos modelo y
representati·os de los existentes en zumo de naranja, son: dos íla·onas agliconas ,quercetina y
kaempíerol,, una íla·anona aglicona ,naringenina,, una íla·ona- O-glicosido ,rutina, y dos
íla·anonas–O-glicosidos ,hesperidina y naringina,. Ll sistema deri·atizante presenta una intensa senal
íluorescente, la cual se monitoriza a las longitudes de onda de excitación y emisión de 585 y 625 nm,
respecti·amente. Lsta senal disminuye al interaccionar el sistema con los analitos, siendo la
disminución proporcional a la concentración de los mismos. Los inter·alos dinamicos de las
calibraciones y los límites de detección, obtenidos con disoluciones estandar de los íla·onoides y
expresados en ng mL
-1
son: 12,2 – 4000, 3,¯ ,quercetina,, 3,5 – 1000, 1,0 ,kaempíerol,, 6,¯ – 1000, 2,0
,naringenina,, 5,0 – 800, 1,5 ,rutina,, 10,1 – 1000, 3,0 ,hesperidina,, y 1¯,8 – 800, 1,8 ,naringina,. Los
coeíicientes de regresión lineal íueron mayores de 0.993 en todos los casos. La precisión de método,
expresada como des·iación estandar relati·a ,°DLR,, íue estudiada a dos ni·eles de concentración
para cada analito, obteniendo ·alores comprendidos entre 2,8 y 6,2 °. La utilidad practica de método
propuesto se ha demostrado mediante su aplicación al analisis de muestras de zumos de naranja, las
cuales íueron diluidas y directamente inyectadas en el sistema cromatograíico. Los porcentajes de
recuperación obtenidos estan comprendidos entre 86,9 y 10¯,0 °.

- 53 -

P-1
DETERMINATION OF ANTIOXIDANT ADDITIVES IN FOODSTUFFS BY
DIRECT MEASUREMENT OF GOLD NANOPARTICLE FORMATION USING
RESONANCE LIGHT SCATTERING DETECTION

A. Andreu-Navarro, J.M. Fernández-Romero, A. Gómez-Hens
Department of Analytical Chemistry
University of Córdoba
Campus of Rabanales
Annex to Marie Curie building
14071-Córdoba. Spain
E-mail: qa1gohea@uco.es
Web: http://www.uco.es/FQM-303/

The capability of antioxidant compounds to reduce gold(III) to gold nanoparticles (AuNPs) has
been kinetically studied in the presence of cetyltrimethylammonium bromide using stopped-flow mixing
technique and resonance light scattering as detection system. This study has given rise to a simple and
rapid method for the determination of several synthetic and natural antioxidant used as additives in
foodstuff samples. The formation of AuNPs was monitored by measuring the initial reaction-rate of the
system in about 5 s, using an integration time of 0.1 s. Dynamic ranges of the calibration graphs and
detection limits, obtained with standard solutions of the analytes, were (mol L
-1
): gallic acid (0.04 –
0.59, 0.01), propyl gallate (0.04 – 1.41, 0.01), octyl gallate (0.03 – 0.35, 0.08), dodecyl gallate (0.02 –
0.30, 0.007), butylated hydroxyanisol (0.07 – 0.39, 0.009), butylated hydroxytoluene (0.04 – 0.32, 0.01),
ascorbic acid (0.11 – 1.72, 0.03) and sodium citrate (0.07 – 1.29, 0.02). The regression coefficients were
higher than 0.994 in all instances. The precision of the method, expressed as RSD%, was established at
two concentration levels of each analyte, with values ranging between 0.6 and 4.8 %. The practical
usefulness of the developed method was demonstrated by the determination of several antioxidant
additives in foodstuff samples, which were extracted, appropriately diluted and assayed, obtaining
recoveries between 95.4 and 99.5 %. The results obtained were validated using two reference methods.




FLUORIMETRIC DETERMINATION OF POLYPHENOLS USING A
LONG- WAVELENGTH-FLUOROPHOR AND LACCASE
IMMOBILISED ON GOLD NANOPARTICLES
Álvaro Andreu-Navarro, Juan Manuel Fernández-Romero, Agustina
Gómez- Hens
Department of Analytical Chemistry. Institute of Fine Chemistry and
Nanochemistry (IUQFN- UCO) Campus de Rabanales. Marie Curie
Building (Annex) University of Córdoba E-14071- Córdoba, Spain.
Phone: 34- 957218645, Fax: 34-957218644, email: qa1feroj@uco.es web:
http://www.uco.es/investiga/grupos/FQM-303
A new method for the determination of polyphenols based on their
competitive redox interaction with a long-wavelength-fluorophor (LWF)
in the presence of the enzyme lacasse immobilised on gold nanoparticles
(AuNPs) is presented. The biocatalyst causes a fluorescence inhibition of
the LWF, but their effect is delayed in the presence of polyphenols. The
competitive redox reaction has been kinetically studied using stopped-
flow mixing technique and fluorimetry as detection system. The behaviour
of several polyphenols (phenol, gallic acid, catechol, hydroquinone,
resorcinol, pyrogallol and phloroglucinol) on the system has been
comparatively studied and a simple and rapid method for the
determination of these compounds has been developed. Initial-rate and
induction time measurements have been used as analytical parameter
using Cardio Green (ex= 764 nm, em= 806 nm) as LWF. Dynamic
ranges of the calibration graphs and detection.