Professional Documents
Culture Documents
UNIVERSITAT DE VALNCIA
Servei de Publicacions
2011
I.S.B.N.: 978-84-370-8800-6
Edita: Universitat de Valncia
Servei de Publicacions
C/ Arts Grfiques, 13 baix
46010 Valncia
Spain
Telfon:(0034)963864115
FACULTAT DE QUMICA
DEPARTAMENT DE QUMICA ANALTICA
DESARROLLO DE MTODOS
ANALTICOS PARA EL CONTROL DE
CALIDAD DE SURFACTANTES Y
ADITIVOS EN PRODUCTOS DE
LIMPIEZA Y DE HIGIENE
PERSONAL
Memoria para alcanzar el grado de Doctor
(Doctorado Europeo) presentada por:
Miriam Beneito Cambra
Directores:
Dr. Guillermo Ramis Ramos
Dr. Jos Manuel Herrero Martnez
Valencia, 2011
II
Certifican
Que la presente memoria, que lleva por ttulo Desarrollo de mtodos analticos
para el control de calidad de surfactantes y aditivos en productos de limpieza y
de higiene personal constituye la Tesis Doctoral de Da. Miriam Beneito
Cambra.
Asimismo, certifican haber dirigido y supervisado tanto los distintos aspectos del
trabajo, como su redaccin.
III
IV
VI
A mi familia
VII
VIII
AGRADECIMIENTOS
La realizacin de esta Tesis Doctoral no habr sido posible sin la ayuda y
el apoyo de un gran nmero de personas:
En primer lugar, quisiera agradecer a mis Directores, el Dr. Guillermo
Ramis Ramos y al Dr. Jos Manuel Herrero Martnez, por su ayuda y dedicacin.
Su confianza, apoyo y paciencia han sido fundamentales para poder llevar a cabo
este proyecto.
A si mismo, quisiera dar las gracias al Dr. Ernesto F. Sim Alfonso, por su
contribucin al desarrollo de mi trabajo y por haber estado dispuesto a apoyarme
cuando lo he necesitado.
Tambin quisiera agradecer a Da. Marisa Caellas y D. Miguel ngel
Chamorro de Qumicas Oro por esta oportunidad que se me ha brindado y la
paciencia durante estos aos.
Cmo no tambin agradecer a todos aquellos que, de un modo u otro
hicieron posible mi estancia en la Universidad de Viena, y principalmente al Dr.
Wolfgang Lindner y Dr. Michael Lmmerhofer, DANKE SCHN.
Por otro lado, agradecer a mis compaeros del Laboratorio 10, con los que
he compartido tantas cosas. cosas buenas, otras no tan buenas, agobios,
preocupaciones, trabajo y momentos de diversin (os acordis de Keops?):
Aarn, Alex, Amparo, Anna, Claudia, Cristina, Elisa, Enrique, Hugo, Isabel,
Ivn, Laura, Mara N., Mara V., Pascuali, Patty, Pietro, Roberto, Sandra,
Tamara, Victoria, Virginia y Yanelis. A la Dra. M Jess (MariJesu!!!!! Que ja
casi ho tinc, no masfixies!!! Moltes grcies per tot, pels consells, xarradetes i
espentes quan fan falta). Todos y cada uno, estis muy presentes, muchas gracias
por todo. A todos los dems compaeros y profesores del Departamento, con los
IX
que ha sido una verdadera satisfaccin relacionarme y trabajar. Tambin dar las
gracias a toda la gente con la que compart muchos momentos en Viena, los
compaeros de laboratorio y otra gente que estuvo ah, en especial al Dr. Xavier
Subirats, moltes grcies per tot, el teu suport, nim i consells son molt valuosos
per a mi.
Tambin agradecer a mis amigos, los de siempre: Amara, Amparo,
Enrique, John, Jose (Teuler), M Mar, Montse, Oscar, Rafa, Ramn, Rebeca,
Vicente y sin olvidar las ltimas incorporaciones (Aharn, Alexis y Nayara),
muchas gracias por estar siempre ah, apoyarme y motivarme para seguir hacia
delante. A todos los amigos de la facultad, que tantas horas hemos compartido en
los pasillos, clases, cafetera y por ah, gracias por entenderme, apoyarme y
aguantarme. Dar las gracias tambin a Pilar y Rosa, que tantos aos
compartiendo el mismo piso, al final une mucho, muchas gracias por todos
aquellos aos y por continuar manteniendo esa amistad.
Y como no, agradecer tambin a mi familia: mis abuelos, tos y primos,
por demostrar siempre tanto inters en mi trabajo y por estar ah, y en especial a
mis padres, quienes siempre han estado a mi lado y me han apoyado en los
momentos difciles, muchas gracias por vuestra ayuda incondicional en todos los
aspectos. Y como no, a mi to Batiste, tu no podies faltar, grcies per tot, ja veus,
tenim algo positiu.
A todos vosotros, y a los que aunque no haya nombrado han sido
partcipes de que todo haya llegado a buen fin, GRACIAS.
XI
NDICE
Captulo I Introduccin....
xx1
I.1. Detergentes....
xx3
I.1.1. Introduccin......
xx3
xx3
I.2. Surfactantes....
x12
I.2.1. Introduccin..
x12
x14
I.2.3. APGs.
x20
x25
I.2.5. AESs..
x29
I.3. Polmeros...
x31
I.3.1. Introduccin..
x31
x34
I.3.3. PVP-NO....
x36
I.3.4. PVP
x37
I.3.5. PVA...
x37
x38
I.4. Enzimas..
x41
I.4.1. Introduccin..
x41
I.4.2. Proteasas....
x43
I.4.3. Amilasas....
x45
I.4.4. Lipasas...
x46
XII
I.4.5. Celulasas........
x47
x49
I.5.1. LC..
x49
x52
I.5.2. CE..
x56
x56
I.5.2.2. EOF
x57
x58
x59
I.5.3. CEC...
x60
x60
x62
x64
x65
I.5.4. MS.
x66
x68
x70
x72
I.5.4.4. Resolucin.
x73
I.5.5. NMR..
x73
x75
x76
x78
XIII
x79
x79
I.5.6.2. LDA...
x80
x83
II.1. Surfactantes..
x85
II.1.1. APGs...
x85
II.1.2. Alcoholes.
x86
II.1.3. AES..
x87
II.2. Polmeros..
x87
II.2.1. PVP-NO...
x87
II.2.2. PVP..
x88
II.2.3. PVA.
x88
II.3. Enzimas....
x89
II.3.1. CZE..
x89
x89
x90
x90
x91
x91
x91
XIV
x93
III.1. Surfactantes.
x95
III.1.1. APGs...
x95
x95
x95
x95
x96
III.1.2. Alcoholes....
x97
x97
x98
x98
x99
III.1.3. AESs...
101
101
101
102
103
106
III.2.1. PVP-NO..
106
106
107
107
108
III.2.2. PVP.
108
108
XV
108
109
110
III.2.3. PVA....
111
111
111
111
114
III.3. Enzimas...
114
III.3.1. CZE.
114
114
116
116
117
118
118
118
119
119
121
122
123
123
124
XVI
124
125
126
126
126
126
127
128
128
129
129
130
132
Results..............
135
137
IV.1. APGs...
139
139
141
141
144
146
XVII
150
152
153
157
159
162
162
164
166
168
169
171
173
179
V.1. AESs.....
181
181
184
186
XVIII
190
193
194
197
IV.1. PVP-NO......
199
199
202
206
208
210
215
VI.2. PVP.....
217
217
219
222
223
226
228
XIX
231
233
235
VI.3. PVA.....
239
239
241
242
244
247
250
254
259
261
261
263
264
266
267
XX
269
269
271
272
276
276
276
278
280
284
284
287
290
295
297
299
299
XXI
301
311
316
316
317
324
329
IX.1. Surfactantes.
331
IX.1.1. APGs..
331
331
333
IX.1.2. Alcoholes....
335
335
IX.1.3. AES....
341
341
XXII
344
IX.2.1. PVP-NO.
344
344
IX.2.2. PVP.
347
347
IX.2.3. PVA....
350
350
IX.3. Enzimas..
354
IX.3.1. CZE....
354
354
356
356
357
357
359
359
XXIII
362
362
362
364
Captulo X Referencias.
367
Abreviaturas.
399
Anexo I
Separation and determination of alkylglycosides by liquid
cromatography with electrospray mass spectrometric detection.
xA3
Anexo II
Study of the fragmentation of D-Glucose and
Alkylmonoglycosides in the presence of sodium
ions in an Ion-Trap Mass Spectrometer.......................................
A13
Anexo III
Chromium(VI) oxide oxidation of non-ethoxilated and
ethoxilated alcohols for determination by electrospray
ionization mass spectrometry..
A31
Anexo IV
Characterization of poly(4-vinylpyridine-1-oxide) by
free-solution capillary electrophoresis and
micellar electrokinetic chromatography..................................
XXIV
A41
Anexo V
Characterization and determination of poly(vinylpyrrolidone)
by complexation with an anionic azo-dye and
nonequilibrium capillary electrophoresis....
A51
Anexo VI
Evaluation of molecular mass and tacticity of polyvinyl alcohol
by non-equilibrium capillary electrophoresis of
polymer-dye equilibrium mixtures..
A61
Anexo VII
Rapid classification of enzymes in cleaning products by hydrolysis,
mass spectrometry and linear discriminant analysis.... A71
Anexo VIII
Enzyme class identification in cleaning products by hydrolysis
followed by derivatization with o-phthaldialdehyde,
HPLC and linear discriminant analysis...
A79
Anexo IX
Photo-polymerized lauryl methacrylate monolithic columns
for CEC using lauroyl peroxide as initiator....
A87
Anexo X
Comparison on photo-initiators for the preparation
of methacrylate monolithic columns
for capilary electrochromatography.....................................
XXV
A99
CAPTULO I
INTRODUCCIN
Captulo I. Introduccin
I.1. Detergentes
I.1.1. Introduccin
El jabn es conocido desde las culturas antiguas (egipcios y sumerios),
siendo usado tanto para lavar la ropa como para el aseo personal. Los jabones se
obtenan mediante saponificacin de grasas vegetales o animales con cenizas
vegetales o minerales como la sosa custica. Hasta el siglo XV, uno de los
principales ncleos de vida social en las ciudades europeas eran los baos
pblicos. En los siglos posteriores, los baos fueron considerados inmorales y el
jabn pas a ser algo a evitar. Se vesta la misma ropa durante semanas y los
malos olores se tapaban con perfumes. En Europa, el jabn no volvi a apreciarse
hasta bien entrado el siglo XVIII, cuando los mdicos se dieron cuenta de la
importancia de la higiene para la salud. Por otro lado, la industrializacin y las
importaciones de grasas baratas facilitaron la fabricacin de jabones a gran escala
[Ramrez-Corrales, 2006].
[www.ambientum.com].
A) Surfactantes
Los surfactantes actan como agentes humectantes del sustrato, modifican
la tensin superficial del agua y emulsionan las partculas de suciedad. En
algunas aplicaciones se usan las propiedades bactericidas de ciertos surfactantes
en formulaciones desinfectantes [Showell, 2006].
Captulo I. Introduccin
D) Agentes antirredeposicin
Estas sustancias impiden que las suciedades separadas de los tejidos
durante el lavado vuelvan a depositarse sobre los mismos. Los agentes
antirredeposicin ms utilizados son la carboximetilcelulosa, y otros derivados
no inicos de la celulosa. Comercialmente tambin se utilizan polmeros
sintticos como PVP, PVA y algunos copolmeros de stos [Salager, 1988].
[Salager, 1988;
F) Agentes blanqueadores
La obtencin de blancura en textiles es quizs una de las propiedades ms
importantes para el consumidor. En el mercado existen dos tipos de agentes
blanqueadores para textiles, ambos con propiedades oxidantes: los hipohaluros,
como el hipoclorito de sodio, y las sales inorgnicas peroxigenadas, como el
perborato de sodio y el perxido de sodio estabilizado con carbonato sdico
(percarbonato). Los agentes blanqueadores oxidantes deben ser intrnsecamente
inestables para cumplir con su funcin (oxidar), paradjicamente, al mismo
tiempo deben ser estables cuando estn almacenados, solos o en la mezcla
detergente. El hipoclorito es un agente blanqueador ms activo y agresivo que el
perborato, siendo particularmente eficaz en la oxidacin de sustancias que
contienen nitrgeno. Adems de poseer una accin blanqueadora (an a baja
Captulo I. Introduccin
el
percarbonato
carbonato
de
sodio
peroxihidrato
G) Blanqueantes pticos
El grado de blancura obtenido mediante agentes blanqueadores se puede
exaltar mediane el blanqueado ptico. Los blanqueadores pticos son colorantes
orgnicos poliaromticos que absorben luz ultravioleta y emiten una luz azulada
en el visible mediante fluorescencia. A la luz del sol, aaden un tono azulado que
compensa el tono amarillento de los tejidos, por lo que mejora la blancura o
profundidad de los colores. Segn la funcin a la que se destinen, poseen
grupos polares ms o menos importantes para adsorberse sobre fibras hidroflicas
como el algodn, o hidrfobas como el polister. Se han desarrollado agentes
fluorescentes solubles en agua y resistentes al hipoclorito u otros blanqueadores,
gracias a que los tomos de nitrgeno de dichas sustancias estn situados en
estructuras altamente aromticas de tipo benzotriazol o benzofurano
1988].
[Salager,
H) Suavizantes
Despus de un lavado con detergentes sintticos formulados con agentes
secuestrantes, el textil seco presenta una superficie que no es agradable al
contacto con la piel. El residuo de los surfactantes sintticos adsorbidos aumenta
la carga esttica de las fibras, y la ausencia de sustancias con accin lubricante
vuelve al textil relativamente rgido. Los agentes suavizantes contrarrestan
ambos fenmenos, ya que por una parte reducen la carga esttica, y por otra
depositan una capa lubricante. Muchos surfactantes catinicos producen estos
efectos, pero son incompatibles con los surfactantes aninicos utilizados en la
mayora de las formulaciones, por lo que deben usarse por separado. Los mejores
suavizantes de este tipo son las sales de alquil amonio cuaternario y de
imidazolio. Existe una tendencia hacia la produccin de formulaciones que
contengan agentes suavizantes compatibles con los agentes limpiadores. Estos
suavizantes son surfactantes con cierto carcter catinico que se absorben sobre
las fibras textiles, siendo a la vez compatibles con surfactantes aninicos
comnmente empleados. Para este fin se usan surfactantes no inicos con un
grupo nitrogenado, o tambin algunos surfactantes anfteros, normalmente
conteniendo un grupo amido-amina (que tambin actan como dispersantes de
jabones de calcio) [Salager, 1988; Showell, 2006].
I) Enzimas
Se utilizan uso en las formulaciones de detergentes para eliminar las
manchas, gracias a la rotura cataltica de componentes especficos de las mismas.
Las proteasas, encargadas de la degradacin de las protenas, son las ms
comunes en la mayora de los detergentes. Sin embargo, tambin se emplean
otras enzimas como las amilasas (degradan el almidn), lipasas (degradan
lpidos) y celulasas (degradan la celulosa). Estas enzimas son capaces de
Captulo I. Introduccin
J) Polmeros
El uso de polmeros ha ido aumentando a medida que las formulaciones de
detergentes han evolucionado. Los polmeros han venido a sustituir a los fosfatos
que fueron cayendo en desuso a causa de su elevado impacto ambiental. Se han
usado principalmente polmeros de tipo poliacrilato, con el fin de remplazar a
agentes secuestrantes como el tripolifosfato de sodio. La carboximetilcelulosa
fue tambin uno de los primeros polmeros utilizados para este fin. Los xitos
obtenidos con los polmeros para otros propsitos ha impulsado la aparicin de
un gran nmero de stos
[Zini, 1999].
K) Espesantes
A menudo es conveniente modificar la reologa, es decir, la consistencia
de la formulacin del detergente en condiciones dinmicas, lo que tiene
importantes consecuencias prcticas. As por ejemplo, los lavavajillas contienen
espesantes que ayudan a mantener en suspensin el fosfato y otras sustancias que
de otra forma quedaran separadas de la fase lquida. El espesado puede
conseguirse a partir de electrolitos inorgnicos, como por ejemplo, NaCl, arcillas
como la laponita o hectorita, o polmeros de un elevado peso molecular como la
carboximetilcelulosa y las gomas de guar o xantana. Existen polmeros
L) Perfumes
En un sentido tcnico los perfumes no aaden un mayor poder detergente
a los formulados, sin embargo, desde el punto de vista del consumidor
representan un factor importante, ya que segn el aroma parece que el detergente
funcione mejor. El olor es una caracterstica que debe ser cuidadosamente tratada
en la formulacin de los detergentes para la aceptacin del producto por parte de
los consumidores. Los perfumes son complejas mezclas de compuestos
orgnicos, por ejemplo, el perfume de un detergente puede estar compuesto por
30, 50 o incluso ms de 100 compuestos. Esta compleja naturaleza puede dar
lugar a complejas interacciones con el resto de componentes del detergente, que
pueden afectar tanto al perfume como a las sustancias activas. En detergentes de
uso domstico, particularmente para lavavajillas y desinfectantes, se incorporan
perfumes, la mayora de los cuales son terpenos, cuyo esqueleto est compuesto
de 2, 3 ms unidades del isopreno (2-metil-butadieno)
2006].
10
Captulo I. Introduccin
M) Disolventes
La seleccin de los disolventes empleados en las formulaciones de
detergentes depende de la naturaleza de las sustancias activas presentes en los
formulados, de la aplicacin final del detergente y del factor econmico. El agua
es el disolvente ms ampliamente empleado en muchas de las formulaciones de
detergentes para uso domstico e industrial. Sin embargo, muchas de las
sustancias activas comnmente empleadas en formulacin de detergentes
presentan una limitada solubilidad en agua, por lo que se requiere la adicin de
un co-solvente (como el etanol) o de un hidrtopo (como el cumeno sulfonato)
[Showell, 2006].
N) Hidrtopos
En los detergentes lquidos, en ocasiones es necesario incluir hidrtopos
para garantizar la estabilidad del detergente en un amplio intervalo de
temperaturas. Los hidrtropos son sustancias hidroflicas con un grupo apolar,
cuya funcin es aumentar la solubilidad de los surfactantes en formulaciones
lquidas. Los hidrtropos no tienen propiedades surfactantes por ellos mismos,
pero actan como co-solubilizadores a alta concentracin. Los ms utilizados son
los sulfonatos de tolueno, etilbenceno y xileno [Salager, 1988; Showell, 2006].
O) Bactericidas
Ciertas frmulas desinfectantes contienen bactericidas, los cuales pueden
ser surfactantes anfteros que actan tambin como agentes dispersantes de
jabones de calcio. Tambin se usan compuestos catinicos que presentan un
efecto suavizante por sus propiedades antiestticas. Los desinfectantes pueden
tambin contener productos clorados bactericidas y sustancias con propiedades
desodorantes
[Salager, 1988].
11
ms diluidos, donde el agua constituye una gran parte del volumen total del
producto, es normal que la formulacin incluya un agente antibacteriano
(normalmente un bacteriosttico) que alargue el tiempo de vida del producto
[Watson, 2006].
P) Agentes anticorrosin
En las formulaciones de detergentes tambin se pueden encontrar agentes
anticorrosin, con el objetivo de proteger las partes metlicas de las mquinas o
sistemas de lavado. Generalmente, se usa el silicato de sodio que adems posee
un papel secundario como builder [Salager, 1988].
I.2. Surfactantes
I.2.1. Introduccin
La palabra surfactante deriva de la contraccin de los trminos surfaceactive-agent (superficie-activo-agente) y engloba a una serie de especies
qumicas capaces de modificar las propiedades de las interfases de los lquidos
(acuoso o no acuoso) en los que estn presentes. Las propiedades caractersticas
de estas molculas residen en su carcter anfiflico, esto es, de la presencia de
una parte hidroflica y otra hidrofbica en la molcula. Los surfactantes se
concentran en la superficie libre del lquido y en las interfases entre fases
inmiscibles, disminuyendo la tensin superficial. El cambio en la tensin
superficial afecta a la capacidad para formar emulsiones, a la mojabilidad, al
poder dispersante, a la detergencia y a las propiedades solubilizantes.
Los surfactantes poseen una estructura molecular tpica, esencialmente
lineal y asimtrica, con dos zonas, una hidrofbica y la otra hidroflica. La parte
hidrfobica es una cadena aliftica, lineal o ramificada, conteniendo en general
12
Captulo I. Introduccin
13
A) Aninicos
Se caracterizan por tener un grupo hidrfilo cargado negativamente, es
decir, el grupo hidrfilo tiene carcter cido y forma fcilmente un anin. Los
ms antiguos y conocidos son los jabones. Los surfactantes aninicos son los ms
utilizados y han sido considerados como el caballo de batalla en el mundo de
los detergentes. En consecuencia, su produccin es elevada, siendo adems muy
econmicos. Suelen distinguirse las siguientes familias: ABS, AS, AES, APES,
AOS, alquil sulfonatos, -sulfonatos de cidos grasos (inicos y steres de
alquilo), mono- y di-alquil sulfosuccinatos y sulfonatos derivados del petrleo
(Fig. I.1).
Los surfactantes aninicos son especialmente beneficiosos en cuanto a su
accin limpiadora, que se apoya en el hecho de que muchas superficies se
encuentran cargadas negativamente, por lo que no pueden quedarse adsorbidos
sobre las mismas, impidiendo as la redeposicin de sustancias indeseables. En
funcin de la naturaleza del grupo funcional que presenta la carga, muestran una
resistencia variable hacia la hidrlisis. As por ejemplo, los sulfatos se hidrolizan
con facilidad, mientras que los sulfonatos son muy estables. Algunos surfactantes
aninicos, presentan la propiedad de generar fases acuosas viscosas, pudiendo ser
empleados como espesantes. Una limitacin de los surfactantes aninicos es su
tendencia a precipitar en presencia de iones calcio y magnesio, que abundan en
las aguas duras, si bien, los AESs son mucho menos sensibles a los cationes
14
Captulo I. Introduccin
alcalinos que los ASs. Por otro lado, la baja solubilidad y las particulares
propiedades de las interfaces de los precipitados de sulfato de magnesio son
explotadas de forma positiva a la hora de optimizar el rendimiento de los
detergentes.
CH3(CH2)m
CH3(CH2)nCOO- Na+
n + 1 = 10 18
CH3(CH2)n
Jabones
n + m = 7 11
ABS
CH3(CH2)nOSO3 Na+
n + 1 = 12 18
CH3(CH2)n(OCH2CH2)mOSO3 Na+
AS
SO3 Na+
CH
AES
CH3(CH2)nCH
(OCH2CH2)mOSO3 Na+
CH(CH2)mSO3 Na+
m + n = 9 15
m = 1 8 R: cadena alqulica
AOS
APES
ROOCCH
-
CH3(CH2)nSO3 Na+
Alquil sulfonatos
Alquil sulfosuccinatos
B) Catinicos
Los surfactantes catinicos tienen el grupo hidrfilo de carcter bsico.
Suelen agruparse en derivados grasos de amida, amidoaminas, imidazolinas,
derivados del petrleo, nitrilos cclicos alifticos, aromticos, compuestos no
nitrogenados, polimricos catinicos y xidos de amina. En la Fig. I.2 se muestra
la estructura y nomenclatura de las principales familias de surfactantes
catinicos.
15
CH3
CH2NH+
NH+
(CH2)nCH3
(CH2)nCH3
CH3
CH3
n + 1 = 7 18
n + 1 = 7 18
Sales de alquilbencildimetilamonio
Sales de alquilfenildimetilamonio
CH3
N+
(CH2)nCH3
R1
N+
CH3
n + 1 = 12 14
CH3
CH3
R1
N+
CH3
R2
la mayora de las superficies carga negativa, los cationes se retienen sobre ellas
en lugar de solubilizar la suciedad adherida. Sin embargo, y debido a estas
mismas propiedades, poseen numerosas aplicaciones especializadas. Por
ejemplo, las aminas y los compuestos cuaternarios inhiben el crecimiento de
microorganismos como bacterias y algas. Adems las aminas grasas primarias y
las aminopropilaminas grasas se utilizan como inhibidores de la corrosin, y en
la limpieza de metales, cuando se utiliza HCl para disolver el xido. La amina se
orienta en la interfase entre el metal y la disolucin cida, con las colas
hidrfobas comprimidas entre s, formando una capa protectora de una o dos
molculas de espesor. Esta capa es tan cerrada que evita el ataque del metal
limpio por parte del exceso de cido. Otra aplicacin ms de los compuestos
grasos nitrogenados, y que depende de la actividad de la superficie y orientacin
16
Captulo I. Introduccin
C) No inicos
En esta clase de surfactantes, el grupo hidrfilo no es capaz de ionizarse y
formar sales. Los surfactantes no inicos son especialmente tiles por su baja
susceptibilidad a los iones Ca2+ y Mg2+ del agua dura. Aprovechando su
compatibilidad con especies inicas, se suelen mezclar con surfactantes
aninicos, dando lugar a asociaciones beneficiosas. Por ejemplo, los surfactantes
no inicos ayudan a solubilizar las sales de calcio o magnesio de los surfactantes
aninicos. El balance entre la parte hidroflica e hidrofbica en estos surfactantes
se obtiene teniendo en consideracin la cantidad y naturaleza de las unidades
polares y la parte hidrofbica de la molcula (longitud de la cadena
hidrocarbonada). La parte hidrfila de la molcula es casi siempre una cadena de
unidades de EO. Los grupos ter le proporcionan la polaridad necesaria para
garantizar su solubilidad en agua por aceptacin de puentes de hidrgeno. Los
FAEs son los surfactantes no inicos ms empleados en productos de limpieza,
cosmticos, herbicidas, etc. Los APEs, principalmente OPEs y NPEs, tambin
poseen una cadena de unidades de EO, pero a diferencia de los FAEs, absorben
en el UV. La aplicacin de los APEs en detergentes est sometida a restricciones
legales, debido a la difcil eliminacin biolgica (escasa biodegradabilidad) de
los metabolitos ms hidrfobos, concretamente, alquifenoles no etoxilados y
monoetoxilados. Los FAEs lineales se biodegradan ms rpidamente que los
APEs. Adems, tienen mejores propiedades de detergencia que los ABS sobre
17
CH3(CH2)nO(CH2CH2O)mH
FAEs
(CH2CH2O)mH
m = 2 100
APEs
O
(CH2CH2O)nH
RCHN
(CH2CH2O)mH
R: cadena alqulica
FAAs
HOH2C
HO
HO
O
OH
HOH2C
O
O
HO
(CH2)nCH3
OH
n = 8 18 m = 0 3
APGs
Fig. I.3. Estructura y nomenclatura de las principales familias de
surfactantes no inicos.
18
Captulo I. Introduccin
D) Anfotricos
Finalmente las molculas que tienen simultneamente grupos con carcter
cido y bsico son surfactantes anfteros o iones dobles. Son compuestos con
una estructura con una carga positiva y otra negativa simultneamente sobre la
misma molcula. Algunos de estos compuestos tienen grupos cidos o bsicos
dbiles, por lo que pueden comportarse como aninicos o catinicos en funcin
del pH. Usualmente se emplean junto con otros surfactantes (aninicos o
catinicos) para resaltar las propiedades deseadas, como puede ser la detergencia
o la formacin de espuma. Son especialmente utilizados en la formulacin de
productos de aseo personal (gel de bao, champs, etc.) por su suavidad y
compatibilidad con la piel, siendo menos irritantes que los surfactantes catinicos
y aninicos. La formulacin de estos productos es complicada por la posible
precipitacin del surfactante anftero cuando el pH est prximo a su punto
isoelctrico. Pueden utilizarse, junto con NaOH, en limpiadores alcalinos para
superficies grasas, y como limpiadores cidos junto con HCl para superficies
oxidadas, debido a que son estables y funcionales en un amplio intervalo de pH.
Un nmero importante de surfactantes anfteros son compuestos naturales
ampliamente conocidos, como por ejemplo la lecitina. Una familia adicional de
surfactantes anfteros que presentan un grupo amonio cuaternario son las
alquilbetanas (Fig. I.4).
C17H35COOCH2
C17H35COOCH2 O
CH3
CH3
R
CH2OPCH2CH2N+CH3
O-
N+CH2COOCH3
CH3
Lecitina
R: cadena alqulica
Alquil betanas
19
I.2.3. APGs
Los APGs son surfactantes no inicos obtenidos a partir de materiales
renovables (glucosa y cidos grasos)
CH2OH
HOHC
HOHC
5
4
OH
H
3
HO
H
OH
HO
OCnH2n+1
3
H
H
OH
Alquil--D-glucofuransido
OH
OCnH2n+1
H
OCnH2n+1
CH2OH
H
4
OH
Alquil--D-glucofuransido
5
4
H
OH
OCnH2n+1
1
H
H
OH
CH2OH
OH
Alquil--D-glucopiransido
Alquil--D-glucopiransido
Captulo I. Introduccin
Cambra, 2007]).
A) Propiedades y aplicaciones
Los APGs se sintetizan controlando las condiciones para obtener
principalmente estructuras donde la cadena glucosdica presente un DP
comprendido entre 1,2 y 1,7 unidades, y cadenas alqulicas que se hallen en el
intervalo de 8 a 14 tomos de carbono
[Willing, 2006].
Se obtienen as mezclas
21
etoxilados
[Willing, 2006],
B) Mtodos de anlisis
Dado que los APGs se estn convirtiendo en unos surfactantes cada vez
ms interesantes por los diversos aspectos comentados anteriormente, tiene
inters el desarrollo de mtodos para su caracterizacin y cuantificacin
2005].
[Thiele,
valoracin potenciomtrica
[Kroh, 1999].
[Kim, 2001]
[Buchmann,
e hidrlisis enzimtica
[Meissner,
[Steber,
alta temperatura, o bien, con una sililacin previa [Rybinski, 1998; Buchmann, 1996A; Spilker, 1996; Billian, 1998].
[Billian,
[Hbner, 2006].
Captulo I. Introduccin
fotomtrica
[Kramer, 1992],
refractomtrica
[Klaffke, 1998],
ELSD
[Buschmann,
o por MS
Se ha descrito el
[Lafosse, 1992],
anillo.
Los
alquilglucopiransidos
pueden
distinguirse
de
los
Cancilla, 1999; Harvey, 1997; Harvey, 1994; Kovik, 1995; Penn, 1996; Asam, 1997],
las que ms informacin permiten obtener son aqullas que emplean iones sodio
y calcio
La fragmentacin de los
[Zaia, 2004],
y tambin
23
nomenclatura de Domon-Costello
la rotura de los
[Asam,
[Harvey, 2000-A; Harvey, 2000-B; Harvey, 2001; Cancilla, 1999; Asam, 1997;
CH2OH
5
HO
Y0
5
4
OH
HO
CH2OH
0,2X
OH
Z0
2
OH
0,2A
B1
C1
CnH2n+1
O
1
Terminacin reductora
CnH2n+1
OH
y ion cloruro
[Carlesso, 2000].
fluorodesoxiglucosa
24
Captulo I. Introduccin
[March, 2005].
Berman y colaboradores utilizaron el anlisis multivariante de los datos de TOFSIMS para distinguir entre varias furanosas, as como entre varias piranosas
[Berman, 2006].
[Kovik, 2001].
[Sad, 2007].
Los alcoholes
A) Propiedades y aplicaciones
En los ltimos aos, los surfactantes basados en alcoholes grasos han
ganado importancia en el mercado de detergentes debido a sus excelentes
propiedades de lavado y su superior biodegrabilidad. Los alcoholes con una
cadena hidrocarbonada de entre 10 y 18 carbonos (alcoholes grasos) son potentes
feromonas, mientras que los de mayor masa molecular son componentes
esenciales en las ceras de las plantas
[Bianchi, 1995].
La importancia industrial de
[Marcomini, 1996;
26
Captulo I. Introduccin
[Cheremisinoff,
2003].
B) Mtodos de anlisis
Los cellosolves y los alcoholes grasos con una cadena igual o menor a 26
tomos de carbono se han determinado con xito mediante GC [Nichols, 2006], sin
embargo, los FAEs con m > 4, no pueden determinarse por GC debido a su
limitada volatilidad
El principal
27
y el ELSD
cromognicos
y fluorognicos
[Marcomini, 1996; Schmitt, 1990; Kiewiet, 1995; Lemr, 1994; Zanette, 1996; Lemr, 1996;
Sun, 1997; Hoffman, 2004-A; Lemr, 2003; Okada, 1991; Okada, 1992; Hoffman, 2004-B;
Bachus, 2003; Desbne, 2005; Heinig, 1996-A; Mic-Tormos, 2008-A; Mic-Tormos,
2008-B; Mic-Tormos, 2009; Zu, 2010].
Heinig, 1996-A; Zu, 2010; Dunphy, 2001; Cassani, 2004; Sparham, 2005].
Sin
28
Captulo I. Introduccin
[Mic-
I.2.5. AESs
Los AESs son una clase de surfactantes aninicos ampliamente utilizados
en productos de limpieza e higiene corporal
[Fiedler, 1989].
Se obtienen por
CH 3 (CH 2 ) n -1 (OCH 2 CH 2 ) m OSO3que se puede abreviar como CnEmS, donde n adopta valores entre 12 y 16,
mientras que m suele oscilar entre 0 y 3, el valor medio de EO se representa
como m .
A) Propiedades y aplicaciones
Las disoluciones acuosas de sulfatos muestran un comportamiento
especial, debido a que su viscosidad, primero aumenta por adicin de electrolitos
como el cloruro de sodio en bajas concentraciones, disminuyendo con adiciones
posteriores. La mxima viscosidad, en cuanto a la concentracin de la sal a
aadir, depende de la estructura del ter sulfato. En comparacin con los ASs, los
correspondientes AESs son ms solubles en agua y muestran mejor la tolerancia
a la presencia de iones Ca2+ y Mg2+. La introduccin de una cadena de EO en el
29
B) Mtodos de anlisis
Las
principales
caractersticas
de
estos
surfactantes
(viscosidad,
Por lo
la
y la
[Smedes, 1982-
el sulfonato de naftaleno
el sulfonato
Captulo I. Introduccin
hidrlisis de los analitos con cidos diluidos para obtener sus correspondientes
alcoholes y alcoholes etoxilados, seguida de derivatizacin a trimetil sililteres
[Sones, 1979; Kirby, 1975].
I.3. Polmeros
I.3.1. Introduccin
Un polmero es una macromolcula constituida por la unin repetida de
pequeas unidades (monmeros) a travs de enlaces covalentes. Ejemplos de
polmeros de origen natural son las protenas (seda, enzimas, colgeno), los
polisacridos (almidn, celulosa) y los cidos nucleicos, los cuales cumplen
funciones especficas en los seres vivos. Dentro de los polmeros sintticos, el
ms simple es el polietileno, siendo el etileno el monmero a partir del cual se
forma. La unidad estructural que se repite a lo largo de la cadena polimrica se
denomina unidad repetitiva, y la reaccin en la cual los monmeros se unen entre
s para formar el polmero se denomina polimerizacin. Los polmeros consisten
en mezclas de molculas de distintas longitudes de cadena, y por ello se habla de
la MW promedia.
31
al
azar
(ABBBBABABBAAABBA),
de
forma
alternada
32
Captulo I. Introduccin
R H
Dada m
R H
R H
Dada r
R H
R H
n
Fig. I.7. Estructura de los polmeros isotcticos (A), sindiotcticos (B) y
atcticos (C).
33
[Zini,
1999].
A) Agentes antirredeposicin
El efecto de los polmeros en las operaciones de lavado se conoce desde
los aos 60 del siglo XX. La eliminacin de las manchas de las superficies es una
compleja
combinacin
de
procesos
fsico-qumicos
como
disolucin,
34
Captulo I. Introduccin
B) Agentes secuestrantes
Los agentes secuestrantes enmascaran los cationes divalentes del agua
dura, evitando su interaccin con los surfactantes. Los polmeros tambin
utilizados con este fin suelen ser homopoliacrilatos o copolmeros de acrlico y
maleico [Zini, 1999].
35
[Jger, 1991].
I.3.3. PVP-NO
El PVP-NO es un polmero empleado como DTI en las formulaciones de
detergentes. Durante aos, se us principalmente PVP y sus copolmeros, sin
embargo, la eficacia de estos polmeros para atrapar colorantes se reduce en
presencia de surfactantes aninicos
[Oakes, 2003-A].
[Lee, 1996].
CH2 CH
N+
N
O-
4-vinilpiridina
PVP-NO
36
Captulo I. Introduccin
I.3.4. Poli(vinilpirrolidona)
De la primera polimerizacin de la N-vinilpirrolidona se obtuvo un
polmero soluble, el PVP (Fig. I.9), patentado en 1939. La polimerizacin se
realiza mediante radicales libres en agua o en 2-propanol, utilizando como
iniciador perxido de hidrgeno o un perxido orgnico, respectivamente [Bhler,
2005].
C H C H2
N
O
C H C H2
N
O
n
N-vinilpirrolidona
PVP
37
de potasio en etanol
[Gallardo, 1999].
consecuencia
de
propiedades
tales
como
su
inocuidad,
alta
CH
O
CH2
O
C
CH3
CH
OH
PVAcO
PVA
de
polmeros
sintticos
[Gallardo,
1999;
Cottet,
2005].
38
Captulo I. Introduccin
comportamiento
de
polmeros
en
disolucin,
permiten
estimar
[Bohrisch, 2000].
Gallardo y col. [Gallardo, 1999] separaron por MEKC una fraccin mayoritaria en
metacrilato y otra rica en vinilpirrolidona a partir de copolmeros no inicos.
Tambin, el empleo de MEKC ha proporcionado informacin sobre el progreso
de la sntesis, naturaleza y composicin de copolmeros inicos
[Aguilar, 2002].
Por otro lado, en CGE es posible conseguir la discriminacin por masa molecular
de polmeros inicos mediante un proceso de tamizado molecular, a travs de un
BGE que contiene un polmero no inico, como el PEG
derivados de celulosa, como dextrano u otros polisacridos
[Grosche, 2000]
[Cottet, 2001].
En estos
copolmeros, las cadenas libres (unmeros) presentan una movilidad elevada (en
trminos absolutos), mayor que la movilidad de los polmeros micelizados.
39
Adems, el pico del polmero micelizado es ms ancho que el pico del polmero
libre, lo que se ha atribuido a cierta dispersin en el nmero de agregacin.
Gyorffy y col.
[Gyrffy, 1998]
40
Captulo I. Introduccin
[Karimi,
I.4. Enzimas
I.4.1. Introduccin
Las enzimas aparecieron en el mercado de los productos de limpieza en la
dcada de 1960, para ayudar a eliminar las manchas proteicas. Hoy en da, la
presencia de diferentes enzimas en las formulacioens mejora la detergencia, ya
que facilitan la degradacin de grandes y complejas molculas tales como las
protenas, almidones y grasas. Los productos de reaccin obtenidos tras la
actuacin de las enzimas son ms solubles en las aguas de lavado, por lo que son
arrastrados ms fcilmente por los surfactantes. Por otro lado, las enzimas
tambin ayudan a mantener la blancura y brillo en los tejidos, as como a
41
clarificar los colores, ya que pueden eliminar las fibras desprendidas de los
tejidos (pelusa) [Crutzen, 1999].
La incorporacin de las enzimas a los detergentes en polvo es muy
habitual, siendo menos frecuente en detergentes lquidos, por la mayor dificultad
para preservar su estabilidad. Tampoco suelen usarse en patillas de jabn ni en
lavamanos, debido a su inestabilidad durante los procesos de fabricacin, y por
posibles procesos de irritacin cutnea. Los detergentes enzimticos representan
un 30% del consumo mundial total de detergentes, el 80% del mercado en USA
[Krawczyk, 1996]
42
Captulo I. Introduccin
serian difciles de quitar slo con los detergentes. Tambin cabe mencionar que
al actuar como catalizadores, las enzimas suelen ser muy efectivas a bajas
concentraciones (menos de 0,1% en peso de enzima en el detergente)
[Crutzen,
1999].
[Baas, 1997].
especialmente los ABS, tambin degradan las enzimas, mientras que los
surfactantes no inicos no las desestabilizan
[Bahn, 1987].
Las enzimas se
I.4.2. Proteasas
Las proteasas son las enzimas encontradas mayoritariamente en los
detergentes. Como se ha descrito anteriormente, las proteasas catalizan la rotura
de largas protenas en molculas ms pequeas, como pptidos o aminocidos,
que pueden ser ms fcilmente eliminadas por los surfactantes. Segn la posicin
activa, las proteasas pueden clasificarse en dos grupos:
43
[Sarath, 1989].
Las protenas
[Ballinger, 1998].
44
Captulo I. Introduccin
[Vinther, 1992-A].
I.4.3. Amilasas
Las amilasas aumentan la eficacia de los detergentes ayudando a disolver
las manchas constituidas mayoritariamente por almidn, si bien, la mejora en la
eficacia limpiadora es menor que la obtenida con proteasas. Estas enzimas son amilasas de origen bacteriano (Bacillus licheniformis) que catalizan la hidrlisis
de los enlaces -1,4 glicosdicos de la amilosa y la amilopectina en dextrinas,
oligosacridos y azcares, dando lugar a molculas solubles. Las amilasas
presentan una mayor estabilidad frente a pHs alcalinos y a temperaturas altas que
las proteasas. Para mantener la actividad de las amilasas, el medio debe contener
suficiente concentracin de ion Ca2+, necesaria para estabilizarlas frente a la
desnaturalizacin y al ataque de las proteasas [Crutzen, 1999].
Las amilasas pueden detectarse mediante el uso de tabletas de Phadebas
o productos similares constituidos por almidn entrecruzado, insoluble y
coloreado; en presencia de amilasa se produce su degradacin, con formacin de
dextrinas y liberacin de colorantes solubles. Hasta ahora, las principales
amilasas usadas en detergentes derivan del Bacillus licheniformis, cuya enzima
45
I.4.4. Lipasas
Los triglicridos son los principales constituyentes de las manchas
procedentes de grasas animales y aceites vegetales. Las lipasas empleadas en
detergencia catalizan la hidrlisis de los enlaces ster de los principales
componentes de las grasas, los TAGs. Por lo tanto, las lipasas ayudan a eliminar
las manchas grasas, las cuales se adhieren fuertemente a los tejidos, y adems
evitan su redeposicin durante el lavado. Las lipasas son una buena muestra de la
cooperacin enzima-surfactante. Estas enzimas actan a temperaturas a las cuales
la solubilizacin de las grasas por parte de los detergentes es baja. La reaccin
enzimtica conduce a una mezcla de diglicridos, monoglicridos y glicerol, los
cuales son menos hidrofbicos que los materiales originales, siendo por tanto
ms fcilmente arrastrados por los surfactantes [Crutzen, 1999].
La primera lipasa industrial, Lipolase, de la casa comercial Novo, y
aislada del hongo Humicola lanuginosa, apareci en el mercado en 1988. Gracias
a la ingeniera gentica, la Lipolasa puede ser producida con buenos
rendimientos a partir del inofensivo microorganismo Aspergillus oryzae. En
relacin con la accin limpiadora, la lipasa difiere de proteasas y amilasas, ya
que se necesitan varios lavados para observar sus beneficios
Gormsen, 1991].
[Aaslyng, 1991;
46
Captulo I. Introduccin
la enzima es poco activa cuando el contenido de agua en los tejidos est por
debajo del 5%, o por encima del 80%, siendo mxima con un contenido de agua
del 80%
[Gormsen, 1991].
Ya que
I.4.5. Celulasas
Todas las enzimas consideradas hasta el momento solubilizan las manchas
por la degradacin de los principales constituyentes. Las celulasas ejercen un rol
bastante diferente y proporcionan otros beneficios: suavidad de las fibras,
47
[Christensen, 1987].
[Whalley, 1994].
[Boyce, 1986;
entre las distintas clases de enzimas utilizadas en detergentes. As, las celulasas
pueden obtenerse a partir de Humicola insolens, diversas especies de los gneros
Trichoderma, Thielavia o Melanocarpus, o varias especies de Bacillus, as como
a partir de hongos.
Las celulasas se pueden detectar mediante el uso de mtodos de tincin
basados en el CR o en el azul de tripn. Estos mtodos, bien conocidos en el
mbito de la Bioqumica Analtica
se basan en la
La identificacin de
[Chernoglazov, 1989].
48
Captulo I. Introduccin
[Eriksson,
1985].
I.5.1. LC
La cromatografa lquida es un mtodo fsico de separacin en el cual los
componentes a separar se distribuyen entre dos fases: la fase estacionaria, que
permanece fija, y la fase mvil, que se mueve en una direccin definida. Un
sistema cromatogrfico (Fig. I.11) se compone al menos de un sistema de
bombeo, un dispositivo para la introduccin de la muestra o inyector, una
columna y un detector, y un sistema adecuado para la adquisicin de datos y
control.
Las bombas empleadas en LC pueden ser de dos tipos: bombas isocrticas
o bombas de gradiente. Los mdulos de bombas para gradiente generan,
mediante su control programado, mezclas de composicin variable. Segn el tipo
49
de bomba, las mezclas se preparan a baja o a alta presin, siendo distintas las
caractersticas, ventajas y limitaciones de cada tipo.
Fase mvil
Detector
Inyector
Sistema de
reparto
Residuo
Registro
de datos
Inyeccin de la
muestra
Conc.
A B C
tR
Cromatograma
Captulo I. Introduccin
51
t R ,i t0
t0
(E.I.1)
Captulo I. Introduccin
k inferiores a 0,2 indican poca retencin, preferencia excesiva del soluto por la
fase mvil. Por el contrario, valores de k superiores a 20 indican una retencin
demasiado alta, producida por una preferencia excesiva del soluto por la fase
estacionaria, lo que implica tiempos de anlisis muy largos y en general picos
anchos y de baja altura, difciles de detectar y de medir con precisin, y por
tanto, con LODs mayores de lo deseable.
La capacidad de un sistema cromatogrfico para distinguir entre dos
solutos se expresa mediante el factor de selectividad i,j, que se calcula como el
cociente entre las retenciones relativas de ambos solutos:
i, j =
kj
(E.I.2)
ki
t R ,i t R , j
(E.I.3)
0,5( wi + w j )
t
N = 5,54 R
w1 / 2
(E.I.4)
53
L
H
(E.I.5)
B
+ C u
u
(E.I.6)
Trmino A
BB
54
Captulo I. Introduccin
55
H = A+
B
+ C u
u
u
C u
B
u
A
Uptima
Velocidad lineal promedia
I.5.2. CE
La CE es una tcnica de separacin que se basa en la migracin diferencial
de especies cargadas en el seno de un tubo capilar y en presencia de un campo
elctrico. La fuerza que impulsa el flujo en CE se genera en la misma pared
interna del capilar mediante el fenmeno conocido como electromosis
[Landers,
1994; Li, 1992; Camilleri, 1993; Heiger, 1992; Brown, 1995; Rogan, 1993].
(E.I.7)
(E.I.8)
Ff = 6rv
(E.I.9)
56
Captulo I. Introduccin
(E.I.10)
De esta ecuacin se deduce que las partculas con mayor densidad de carga
elctrica tendrn movilidades mayores. La movilidad se considera positiva
cuando el ion se dirige al ctodo. La movilidad medida en funcin del tiempo de
migracin se conoce como movilidad aparente o efectiva (ap), y difiere de la
intrnseca o electrofortica cuando se observa en presencia de EOF no nulo. La
movilidad aparente corresponde a la suma de las movilidades electrofortica y
electroosmtica, siendo la velocidad total de las partculas cargadas la suma de
sus velocidades electrofortica y electroosmtica:
v = ve + vEOF = apE = (e +EOF)E
(E.I.11)
I.5.2.2. EOF
El EOF es uno de los principales fenmenos que afectan a las
separaciones electroforticas. En contacto con un medio acuoso, la superficie del
capilar de slice tiene generalmente un exceso de carga negativa que es debido a
la ionizacin de los grupos silanol. Para capilares de slice fundida, el EOF se
puede controlar reduciendo o aumentando el nmero de grupos silanol en forma
ionizada. As, el EOF es prcticamente nulo por debajo de pH 3, y aumenta con
el pH. Para la slice se tiene: log Kmedio 5,5.
Los iones que se encuentran en la disolucin tienden a neutralizar la carga
de la superficie del capilar, formando una doble capa elctrica, y creando una
diferencia de potencial residual conocida como potencial zeta. La primera capa, o
capa de adsorcin primaria, est fuertemente retenida, pero sobre ella se
establece una capa difusa en la que predominan los iones del signo contrario al
57
potencial zeta de la superficie. Esta segunda capa est menos retenida, por lo que
puede moverse por aplicacin de una diferencia de potencial, y en su movimiento
arrastra a todo el lquido. En la Fig. I.14 se representa esquemticamente este
fenmeno.
+
+
-
Capa de
adsorcin
primaria
+
-
+
+
+
-
+ +
+ +
-
+
-
+
-
+ +
+
+
+
+
+
+
+
+
-
+
+
+
+
-
+
+
EOF
-
+
-
+
-
+
-
+ -
+
-
+
+
+ +
+
-
+
-
+
+
+
-
+
+
+
-
+ +
+
-
+ + + + +
+
+
+
+
+
+
-
+
+
-
+
-
Capa
difusa
+
+
+ + +
(E.I.12)
(E.I.13)
Captulo I. Introduccin
ap =
l
lL
=
tE tV
(E.I.14)
A) FSCE o CZE
En la FSCE o CZE, el capilar se llena slo con el BGE, y la separacin
tiene lugar gracias a las diferentes movilidades electroforticas de los solutos. La
seleccin del BGE es extremadamente importante, ya que la selectividad de la
separacin depende en gran medida de su naturaleza. Los reactivos utilizados
deben cumplir las siguientes condiciones:
i) Buena capacidad amortiguadora en el intervalo de pH requerido.
ii) Baja absorbancia a la longitud de onda de deteccin.
iii) Baja movilidad electrofortica de sus componentes para minimizar la
corriente.
B) MEKC
La MEKC fue introducida en 1984 por Terabe y col. [Terabe, 1984; Terabe,
1989; Terabe, 1992; Otsuka, 1989],
59
solutos. Las especies no cargadas que interaccionan con las micelas adquieren
movilidad, distinguindose del EOF, y las que no interaccionan se mueven con el
mismo. Se puede aplicar tambin a especies inicas, si bien, en este caso el
mecanismo de separacin es mixto, cromatogrfico y electrofortico a la vez.
I.5.3. CEC
La CEC es una tcnica analtica de separacin en fase lquida que combina
la elevada eficacia de la CZE con la alta selectividad y reproducibilidad
proporcionadas por la HPLC. En CEC, la separacin se lleva a cabo en columnas
capilares rellenas total o parcialmente con una fase estacionaria. Como en CZE,
el EOF es generado por el campo elctrico. Para que se genere EOF debe
asegurarse la presencia de cargas sobre la superficie del relleno de la columna.
Por otro lado, el EOF es el responsable del bombeo en CEC y en otras tcnicas
de electroseparacin. El bombeo mediante el EOF da lugar a un perfil plano de
velocidades en el seno de la fase mvil, a diferencia del perfil parablico que se
obtiene cuando el bombeo es por presin externa, como en HPLC. El perfil plano
del flujo es uno de los factores que permite obtener elevadas eficacias.
En CEC el mecanismo de separacin es doble [Rathore, 1996]. Por un lado,
hay un mecanismo cromatogrfico, ya que se produce un reparto de los solutos
entre una fase mvil y una estacionaria. Por otro lado, los solutos inicos tambin
se separan mediante un mecanismo electrofortico, esto es, en base a las
diferencias de movilidad electrofortica, por lo que la naturaleza del relleno de la
columna determina el EOF e influye sobre la selectividad de la separacin.
60
Captulo I. Introduccin
Capilar
relleno
Fuente de
presin
BGE
[Carney, 1999],
por
por la presencia de las fritas que mantienen la integridad estructural del relleno
[Rebscher, 1994].
61
siendo
62
Captulo I. Introduccin
[Ericson, 1999].
La
Fujimoto,
1995;
Hoegger,
2001].
[Hjertn,
63
se
Zhang,
2003;
Peters,
1998-A;
Peters,
1998-B]
[Merthar,
se preparan mediante
64
Captulo I. Introduccin
[Baeuml, 2002],
ecuacin de Brunauer-Emmet-Teller
[Brunauer,
1938]
y la permeabilidad
65
I.5.4. MS
La MS es una tcnica de aplicacin general, capaz de suministrar
informacin sobre la composicin cualitativa y cuantitativa, tanto de analitos
orgnicos como inorgnicos, en muestras complejas. Los espectros de masas se
obtienen por conversin de los componentes de una muestra en sus respectivos
66
Captulo I. Introduccin
iones en fase gas, que se separan en funcin de su relacin m/z, siendo la seal
analtica la abundancia o intensidad para cada valor m/z.
En la Fig. I.16 se muestra un esquema de los componentes principales de
un espectrmetro de masas.
Muestra
Analizador
Analizador
demasas
masas
de
Fuentede
deiones
iones
Fuente
Sistema
Sistema
devaco
vaco
de
Detector
Detector
Procesador
Procesador
dela
laseal
seal
de
Dispositivo
Dispositivo
delectura
lectura
de
67
68
Captulo I. Introduccin
Gas de secado
Aerosol
69
[Rubinson,
2001].
70
Captulo I. Introduccin
Eyeccin secuencial
Incremento de V
Fig. I.18. Esquema de funcionamiento de una IT.
Entrada
Gas
nebulizador
Nebulizador
Desecho
Vaporizacin
y ionizacin
(ESI)
Gas de
secado
Enfoque
Analizador
Deteccin
(IT)
72
Captulo I. Introduccin
I.5.4.4. Resolucin
La capacidad de un espectrmetro de masas para distinguir entre masas
similares se expresa normalmente en trminos de resolucin, R, que se define
como:
R = m/m
(E.I.15)
I.5.5. NMR
La espectroscopa de NMR fue desarrollada a finales de los aos cuarenta
para estudiar los ncleos atmicos. En 1951, se descubri que la espectroscopa
de resonancia magntica nuclear poda ser utilizada para determinar las
estructuras de los compuestos orgnicos. Esta tcnica espectroscpica puede
utilizarse slo para estudiar ncleos atmicos con un nmero impar de protones o
neutrones (o de ambos). Esta situacin se da en ncleos de una docena de
elementos, entre ellos los de 1H,
13
C,
19
F y
31
73
H0
2
74
(E.I.16)
Captulo I. Introduccin
Tubo con
muestra
Detector
y
Amplificador
Generador de
radiofrecuencia y
ordenador
Imn
superconductor
I.5.5.2. NMR de 1H
Hasta ahora se ha descrito el concepto de resonancia de un ncleo aislado
dentro de un campo magntico, pero los ncleos se encuentran rodeados de
electrones que los protegen parcialmente del campo magntico externo al que se
ven sometidos. Los electrones se mueven generando un pequeo campo
magntico inducido que se opone al campo magntico externo. En cualquier
molcula, la nube electrnica que existe alrededor de cada ncleo acta como
una corriente elctrica en movimiento que, como respuesta al campo magntico
externo, genera una pequea corriente inducida que se opone a dicho campo. El
resultado es que el campo magntico que realmente llega al ncleo es ms dbil
que el campo externo, por tanto, se dice que el ncleo est apantallado. Este
apantallamiento es importante desde el punto de vista experimental, ya que el
campo magntico efectivo (Hef) que siente un protn dentro de una molcula es
siempre menor que el campo externo, y por lo tanto, para que el ncleo entre en
resonancia dicho campo externo debe ser mayor.
Si todos los protones (1H) de una molcula estuvieran apantallados de
igual forma, todos entraran en resonancia con la misma combinacin de
frecuencia y campo magntico. Sin embargo, los protones se hallan dentro de
entornos electrnicos diferentes y, por tanto, diferentemente protegidos o
76
Captulo I. Introduccin
77
13
12
nmero par de neutrones, por tanto, no tiene espn magntico y no puede dar
lugar a seales de NMR. El istopo de
13
13
78
13
C sea
Captulo I. Introduccin
79
I.5.6.2. LDA
En LDA se utiliza un algoritmo que busca funciones o vectores
discriminantes, esto es, combinaciones lineales de las variables manifiestas que
maximizan la varianza entre categoras, a la vez que minimizan las varianzas
intra-categoras. Para construir el modelo, es necesario asignar los objetos del
conjunto de entrenamiento a una categora dada. Para ello, se aade una variable
categrica a la matriz de datos conteniendo tantas categoras como sean
necesarias. El LDA estima los coeficientes a1, a2, , am de la funcin
discriminante lineal, f, que es capaz de predecir la pertenencia de los objetos a
una u otra categora:
f = a1 x1 + a2 x2 + ... + am xm
(E.I.17)
SC D
SCI
(E.I.18)
donde SCD es la suma de cuadrados de las distancias eucldeas entre los objetos
que pertenecen a distintas categoras en la direccin que indica la funcin
discriminante buscada, y SCI es la suma de los cuadrados de las distancias
eucldeas entre los objetos que pertenecen a la misma categora, tambin en la
direccin de la funcin discriminante. A partir de q categoras se obtienen q-1
funciones discriminantes (aunque si el nmero de variables predictoras, N, es
menor que q, se obtendrn N-1 funciones discriminantes). Las funciones
discriminantes se obtienen en orden decreciente de su valor de , y manteniendo
la ortogonalidad entre ellas.
La funcin no est acotada, por lo que vara ampliamente con el
nmero de objetos y con la separacin entre ellos. Por ello, en lugar de
maximizar , se suele minimizar la lambda de Wilks, que se define como:
80
Captulo I. Introduccin
W =
1
SCI
=
1 + ' SCI + SCD
(E.I.19)
81
CAPTULO II
II.1. Surfactantes
II.1.1. APGs
Se propone el desarrollo de mtodos para la separacin y determinacin de
APGs. El estudio de la separacin de mezclas de APGs se llev a cabo mediante
HPLC-MS usando columnas de alquilamida y cianopropilo, con mezclas
ACN/agua como fases mviles. Se optimizaron las condiciones de elucin para
conseguir la resolucin completa de epmeros (- y -) e ismeros de anillo
85
II.1.2. Alcoholes
Se pretende el desarrollo de un procedimiento de derivatizacin que
permita aumentar la sensibilidad de los alcoholes no etoxilados y etoxilados por
ESI-MS-IT. Para ello, los alcoholes primarios sern previamente oxidados a sus
correspondientes cidos carboxlicos con el reactivo de Jones. Los extractos se
infundirn directamente en ESI-MS-IT. El estudio se extender a FAEs y a los
disolventes conocidos como Cellosolves, que al oxidarse proporcionan los
correspondientes cidos etoxi-carboxlicos. Los procedimientos establecidos se
aplicarn a la caracterizacin y determinacin de alcoholes grasos en muestras
complejas, tales como cosmticos y productos de aseo personal, as como a
muestras ambientales como agua de mar.
86
II.1.3. AES
En trabajos anteriores del grupo de investigacin en el seno del cual cual
se ha realizado la presente Tesis, y en el marco de una de sus lneas de
investigacin, se desarrollaron diferentes procedimientos para la determinacin
de FAEs mediante RP-HPLC-UV, previa derivatizacin de los alcoholes con un
anhdrido cclico
Mic-Tormos, 2010].
II.2. Polmeros
II.2.1. PVP-NO
Se estudiar la migracin caracterstica del PVP-NO, tanto en FSCE como
en MEKC. Estas dos tcnicas pueden proporcionar informacin sobre el
comportamiento cualitativo de polmeros en disolucin, as como informacin
cuantitativa sobre muestras que contienen estos aditivos. Se discutir la
naturaleza de las seales obtenidas en base a los estudios de la bibliografa, as
87
como a los nuevos resultados. Para prevenir la adsorcin del polmero sobre las
paredes del capilar se utilizar PEA, que en disoluciones cidas existe como ion
doble de baja masa molecular (sin carga elctrica neta). Este tipo de aditivo
puede usarse en elevadas concentraciones sin causar un aumento significativo de
la conductividad del BGE. Finalmente, se demostrar la utilidad de la FSCE para
determinar este polmero en aditivos comerciales para el lavado de textiles.
II.2.2. PVP
Se pretende desarrollar de un mtodo para la caracterizacin y
determinacin de PVP en productos de limpieza mediante CZE. Dado que la
movilidad electrofortica del PVP es casi cero, se emplearn colorantes azoicos
que formen complejos con el polmero. Estos sistemas pueden interpretarse a la
luz de los estudios de Krylov y col., con aplicacin de los principios en que se
basa la tcnica conocida como NECEEM. A partir de los datos obtenidos y en
base a los estudios de la bibliografa, se buscarn diferentes parmetros
analticos. Posteriormente los procedimientos establecidos se aplicarn a la
caracterizacin y determinacin de PVP en productos de limpieza y formulados
farmacuticos.
II.2.3. PVA
Siguiendo con la determinacin de polmeros no inicos mediante tcnicas
de CE, se pretende el desarrollo de un mtodo para la caracterizacin y
determinacin de este polmero. Dado que la movilidad electrofortica del PVA
es casi cero, al igual que se ha indicado ms arriba para el PVP (apartado II.2.2),
se emplearn colorantes que formen complejos coloreados y cargados con el
PVA, seleccionando los colorantes que mejores resultados proporcionen;
88
II.3. Enzimas
II.3.1. CZE
Se pretende examinar enzimas intactas (protenas nativas) mediante CZE
con deteccin UV, como medio de clasificacin e identificacin de enzimas. Se
estudiarn las cuatro clases principales de enzimas utilizadas en productos de
limpieza: proteasas, amilasas, lipasas y celulasas. Para ello, las enzimas se
precipitarn con acetona, se redisolvern e inyectarn en el equipo de CE. Dado
que la carga y estructura de las protenas dependen en gran medida del pH, y con
el objetivo de reunir ms informacin para la identificacin de enzimas, se
emplearn dos BGEs diferentes, uno a pH cido y otro a bsico. Se utilizar el
ensayo de Bradford para medir las concentraciones de protena en las diferentes
enzimas industriales (seleccionando el BSA como protena de referencia).
89
incluyendo
columnas
monolticas
comerciales
(tanto
90
(es
decir,
relaciones
de
monmeros/porgenos
en
una
mezcla
1,4-butanodiol/1-propanol
como
disolventes
fotografas
SEM.
Adems,
se
evaluarn
sus
prestaciones
91
CAPTULO III
MATERIALES Y MTODOS
III.1. Surfactantes
III.1.1. APGs
13
C), D-glucosa-6,6-d2
13
C) (Sigma-
95
96
III.1.2. Alcoholes
97
98
[Burke, 1999].
disolviendo los alcoholes (50 mg de cada uno) en acetona (25 mL). Alcuotas de
250 L de estas disoluciones se introdujeron en tubos de centrfuga provistos de
tapn de rosca, aadindose 3 mL de acetona y 1 mL del reactivo de Jones, este
ltimo fue aadido gota a gota con agitacin constante a temperatura ambiente.
Tras 5 minutos, se adiciona 0,5 mL de HCl 2M, obtenindose una disolucin de
sales de cromo(III). El exceso de reactivo de Jones, indicado por el color amarillo
de la disolucin, se elimin aadiendo, con agitacin, unos 150 mg de Na2SO3.
Posteriormente, se aadi 4 mL de acetato de etilo. La mezcla se agit para
favorecer la extraccin y luego se centrifug para acelerar la separacin en dos
fases. La fase superior transparente e incolora (con un volumen aproximado de 7
mL) contiene los cidos carboxlicos y etoxicarboxilicos en un medio de acetato
de etilo/acetona (4:3; v/v). Las sales de cromo(III) permanecen en la fase acuosa
(capa inferior) con un volumen de aproximadamente 1 mL. A alcuotas de 2 mL
de la fase orgnica se les aadi 0,2 mL de una disolucin acuosa de butilamina
(0,33 M) que contena TMBA (5,5 mM), este ltimo incorporado como patrn
interno. Esta mezcla se introdujo directamente en el equipo MS.
Para la preparacin de la mezcla industrial FINDET 10/18, y las muestras
de productos cosmticos y de cuidado personal, se tom 1 y 2 g,
respectivamente, se aadi 10 mL de acetona, la mezcla se agit magnticamente
durante 10 min, se centrifug y se tomaron alcuotas del sobrenadante (3 mL).
Como se estudia en la seccin IV.2.1, la reduccin de la cantidad de agua antes
de la adicin del reactivo de Jones es un factor importante para obtener unos
altos rendimientos de la oxidacin. Por este motivo, para muestras con un alto
99
contenido de agua se tomaron volmenes ms pequeos del sobrenadante (0,25 1 mL); estas alcuotas fueron diluidas con acetona hasta un volumen final de 3
mL.
El anlisis de agua de mar se realiz tomando dos muestras de 5 L cada
una. Un volumen de 2 L, tomado de la parte superior de cada contenedor, se pas
a travs de cartuchos de SPE (C18, 500 mg/ 6 mL, 55 m, 140 , Phenomenex,
Torrance, CA, USA) a un flujo de 5 mL min-1. Estos cartuchos se eluyeron con
tres porciones de 2 mL de acetona. El volumen de los eluatos se redujo de 6 a
unos 3 mL por evaporacin en corriente de nitrgeno a temperatura ambiente.
Tras la evaporacin, se efectu la oxidacin y extraccin, tal como se ha descrito
antes. Para construir un blanco de referencia, se tom un tercer volumen de 2 L
de agua de mar, y se procedi a extraccin (SPE) y reduccin del eluato a 3 mL,
como anteriormente se ha indicado. En el procedimiento de oxidacin de este
eluato, el reactivo de Jones fue sustituido por cido sulfrico diluido, siendo el
resto del procedimiento de oxidacin y de extraccin el indicado previamente.
Los espectros MS de los extractos de acetato de etilo/acetona se
registraron en modo NI, y se midieron las abundancias de los iones [M-H]- de los
cidos carboxlicos y etoxicarboxlicos. En los estudios de rendimiento de
oxidacin-extraccin, los espectros tambin se obtuvieron en modo PI, y los
picos de los iones [M+H]+ correspondientes a los alcoholes etoxilados se
compararon con los espectros de los correspondientes cidos etoxicarboxlicos
obtenidos en modo NI. Para obtener espectros en modo PI, alcuotas de 1 mL de
los extractos de acetato de etilo/acetona se acidificaron con 0,1 mL de HCl 2 M
(en lugar de la disolucin de butilamina), esta disolucin se diluy con 1 mL de
ACN, y se infundi en la interfaz ESI del MS. Cuando fue necesario, se emple
el TMBA como patrn interno, ya que proporciona los picos [M-H]- y [M+H]+ en
los modos NI y PI, respectivamente.
100
III.1.3. AESs
101
102
103
104
SAX
Fraccin 4
Fraccin 5
Evaporacin de
disolventes
Neutralizacin
Eluatos de AESs:
Fracciones 4 + 5
combinadas
Evaporacin de
disolventes
105
O
O
R
R
OH
o
+
-
SO3
COO-
H2O
o
SO3
COO-
III.2.1. PVP-NO
106
[Mukherjee, 1971].
107
III.2.2. PVP
108
109
g mL-1 de PVP a las disoluciones de las muestras. Dos bases de detergente, cuya
composicin viene dada en la Tabla III.1, se fortificaron con PVP 60 kDa,
obtenindose una concentracin final de 0,99 y 0,97% (en peso) para las bases I
y II, respectivamente.
Tabla III.1. Composicin de las bases de detergente empleadas en este trabajo
(porcentajes en peso).
Componente
Base I (%)
Base II (%)
AS
AESa
Oleina
3,5
FAEb
11
SDS
1,5
Agua y otrosc
75,5
80,5
III.2.3. PVA
111
112
HO H HO H HO H
HO H
H OH H OH
HO H
H OH HO H
Fig. III.3. Estructuras de las tres posibles tradas en una cadena de PVA:
(A) mm, (B) rm y (C) rr.
Los porcentajes de cada trada se estimaron a partir de las reas relativas de los
tres multipletes que se encuentran dentro del rango de 64-69 ppm [Moritani, 1972;
Wu, 1973; Wu, 1977; Fukae, 2000].
40
61 (1.72)
rr %
100 (1.09) M
30
31 (1.67)
145 (1.04)
15 (1.39)
130 (0.85)
20
49 (1.05)
205 (0.71)
6
2
15
20
25
70
mm %
Fig. III.4. Porcentajes de las triadas rr y mm, estimados por 13C NMR en
las muestras de PVA empleadas en este trabajo (). Los nmeros que se
encuentran junto a los smbolos (), son la MW (kDa) y la relacin rr/mm
(valores entre parntesis). Los otros smbolos corresponden a datos
bibliogrficos
para muestras
Como se observa en esta figura, las muestras de PVA utilizadas en este trabajo
resultaron agrupadas como sigue: un grupo de tres muestras con proporciones
113
rr/mm dentro del rango 1,72 - 1,39, otro grupo de tres muestras dentro del rango
1,09 - 1,04 y finalmente, las dos muestras con mayor MW mostraron los menores
valores de la relacin rr/mm, en concreto 0,85 y 0,71. Para facilitar la discusin
en la seccin IV.3, estos grupos se denominaron en funcin de la relacin rr/mm
como H (alto), M (medio) y L (bajo).
III.3. Enzimas
III.3.1. CZE
114
Clase
Proteasa
Amilasa
Celulasa
Lipasa
a
b
Nombre comercial
Alcalase 2,5 L
Savinase 16L, Type EX
Everlase 16 L, Type EX
Esperase 8.0 L
Polarzyme 12Ta
Bioproteasa L 450
Bioproteasa L 800
Enziprot 450L
Deterzyme L 660
Deterzyme Apy L 560
Properase 1600L
Purafect Prime HA
Properase 4000Da
Excellase 2250Da
Purafect OX 8000Da
Termamyl Ultra 300L
Duramyl 300 L, Type DX
Stainzyme 12L
Enziamilasa
Purastar ST 15000L
Purastar ST 6000Da
Endolase 5000L
Carezyme 4500L
Celluzyme 0,7Ta
Deterzyme CL-5
Puradax HA 400Ea
Puradax EG 7000L
Lipolase 100L, Type EX
Lipex 100L
Lipolase 100Ta
Lipex 100Ta
Fabricantee
Novozymes
Novozymes
Novozymes
Novozymes
Novozymes
Biocon
Biocon
ChemWorld
Enmex
Enmex
Genencor
Genencor
Genencor
Genencor
Genencor
Novozymes
Novozymes
Novozymes
ChemWorld
Genencor
Genencor
Novozymes
Novozymes
Novozymes
Enmex
Genencor
Genencor
Novozymes
Novozymes
Novozymes
Novozymes
Seccin
VII.1; VII.2c; VII.3b,c; VII.4
VII.1; VII.2d; VII.3d
VII.1; VII.2d; VII.3d; VII.4
VII.1; VII.2d; VII.3d
VII.3d
VII.2d; VII.3c
VII.2d; VII.3c
VII.2c; VII.3d
VII.2c; VII.3d
VII.2d; VII.3d
VII.3d
VII.2d; VII.3d
VII.3d
VII.3d
VII.3d
VII.1; VII.2c; VII.3b,c
VII.1; VII.2c; VII.3d; VII.4
VII.1; VII.2d; VII.3c
VII.2c; VII.3c
VII.2d; VII.3d; VII.4
VII.3d
VII.1; VII.2c; VII.3d
VII.1; VII.2c; VII.3b,c
VII.1; VII.2d; VII.3d
VII.2c; VII.3c; VII.4
VII.3d; VII.4
VII.2d; VII.3c
VII.1; VII.2c; VII.3c; VII.4
VII.1; VII.2c; VII.3b,c; VII.4
VII.2c; VII.3c
VII.2d; VII.3d
Muestras suministradas como slidos granulados (el resto de muestras son lquidas).
Se emplean tanto como materia prima industrial como para aditivar las bases de
detergente (Tabla III.3).
115
[Bradford, 1976]
utilizando
116
117
118
[Gimeno-
119
Base I (%)
Base II (%)
AES
10
Olena
10
FAE
10
15
Agua
70
65
120
121
Por ltimo, el conjunto de datos III fue construido a partir de todos los
datos espectrales de los concentrados industriales de enzimas y productos de
limpieza que no se incluyeron en la construccin de los conjuntos anteriores
(Tabla III.2). Este tercer conjunto contena 42 objetos (12 concentrados
industriales de enzimas ms 2 detergentes comerciales 3 hidrolizados de cada
enzima un promedio de las tres infusiones de cada hidrolizado).
[Vandeginste, 1998].
122
[Hurst, 2002].
Los
123
124
estadstico SPSS, donde se adoptaron inicialmente los valores de Fin y Fout, 3,84 y
2,71, respectivamente.
H2N
R1
COOH
+ HS
HN
H
H
Aminocido
OH
O
HO
NAC
125
HN
HO
COOH
R1
O
OPA
OH
Isoindol
126
ProSwift
Monoltica
polimricaa
Chromolith
Monoltica
slice (C18)
Gemini 5 m
Particulada
(C18)
Gemini 3 m
Particulada
(C18)
Kinetex
Particulada
(C18)
2,6
50 4,6
100 4,6
150 4,6
150 3
100 3
Dionex
Merck
Phenomenex
Phenomenex
Phenomenex
127
128
alquilbencenos
conteniendo
tolueno,
etilbenceno,
n-propilbenceno,
n-
los
siguientes
solutos
bsicos: anilina,
4-nitroanilina,
N,N-
129
pH.
5. HCl 0,2 M, hasta observar pH cido a la salida del capilar.
6. Agua nanopura, hasta pH neutro a la salida del capilar, para eliminar los
restos de cido.
7. EtOH hasta olor persistente, con el fin de eliminar el agua y evitar la
hidrlisis del reactivo enlazante (silano-binding) que se adiciona en la
siguiente etapa.
8. Disolucin de reactivo enlazante (silano-binding) al 20% (m/v) en EtOH,
acidulado con cido actico hasta pH 5; el enlazante se pasa a un caudal de
0,25 L min-1 durante 60 min.
9. Acetona, para eliminar el exceso de reactivo enlazante.
Para finalizar, se aplic una corriente de nitrgeno para secar el capilar,
dejndose en estas condiciones durante 24 h hasta completar la reaccin de
condensacin de los grupos silanol con el reactivo enlazante (silano-binding).
Tras este periodo, se retir la fuente de nitrgeno y se sellaron los extremos del
capilar con sendos tapones, con el fin de evitar la hidrlisis de los enlaces
siloxano.
130
131
132
133
RESULTS
CHAPTER IV
IV.1. APGs
[McGregor, 2004].
and in
[Buchmann, 1996-A],
hydrolysis
near-infrared spectrometry
[Kroh, 1999].
[Kim, 2001]
and enzymatic
[Rybinski, 1998;
[Buchmann,
139
[Billian, 1998].
[Hbner, 2006].
To implement
[Kramer, 1992].
or MS detection
[Lafosse, 1992],
and the
separation of APGs with silica, C18 and polyvinylalcohol columns have been
compared
[Czichocki, 2002].
[Eichhorn, 1999]
have used
HPLCMS with an ESI to determine APGs in spiked river and waste waters.
Using a C18 column and gradient elution with ACN/water, the - and -epimers
and the ring isomers were resolved. Alkylglucopyranosides can be further
distinguished by their higher affinity to form [M+NH4]+ adducts with respect to
alkylglucofuranosides. Kuhn and Neubert
[Khn, 2004]
140
response factors was studied. The procedures were applied to the characterization
and determination of APGs in toiletries.
141
-C8G1p
Abundance x 107
1.5
-C8G1p
1.0
-C8G1f
0.5
C8G2
-C8G1f
m/z 315
m/z 477
0
10
20
30
40
50
Time (min)
Fig. IV.1. EIC of Glucopone. The alkylamide column was used. The
mobile phase contained water with 20% ACN in the presence of 40 mM
HAcO/NaAcO buffer of pH 4.7. The flow rate was 0.4 mL min1.
The epimers and ring isomers of C10G1 and C12G1 appeared also resolved at
longer retention times (not shown). Also, a partial separation of the isomers of
the alkyldiglucosides (C8G2) was evidenced (EIC at m/z 477). The identification
of - and -C8G1p was made by injecting solutions of the corresponding
standards. As far as we know, standards of APGf are not available; however, the
two peaks of low intensity which appeared at longer retention times than the and -C8G1p peaks on the m/z 315 trace, were attributed to the corresponding and -C8G1f isomers. Retention should be higher for these isomers as a
consequence
of
the
longer
CH(OH)CH2OH
chain
bound
to
the
142
IV.2), the - and -epimer pairs of C8G1 were not resolved, but the four isomers
of C10G1 were resolved within 20 min. As also shown in Fig. IV.2, the presence
of alkyldiglycosides (C8G2 and C10G2) and alkyltriglycosides (C8G3) with
partial isomer resolution was evidenced.
1.5
C8G1p
Abundance x 106
C10G2
C8G3
1.0
-C10G1p
m/z 639
m/z 505
-C10G1p
C8G2
0.5
C8G1f
-C10G1f
-C10G1f
m/z 315
m/z 343
m/z 477
0
6
8
Time (min)
12
14
16
18
Time (min)
20
Fig. IV.2. EIC of Glucopone. The alkylamide column was used. The
mobile phase contained water with 30% ACN in the presence of 40 mM
HAcO/NaAcO buffer of pH 4.7. The flow rate was 0.2 mL min1.
143
C8G2
m/z 477
2.5
C10G2
m/z 505
Abundance x 107
C12G1p
C12G2
-C14G1f
m/z 533
1.5
-C14G1f
C8G1p
C10G1p
m/z 399
1
m/z 343
0.5
m/z 315
C10G1f
-C14G1p
-C12G1f
-C12G1f
m/z 371
18 20
Time (min)
16
-C14G1p
C8G1f
m/z 399
8
10
14
18
Time (min)
Fig. IV.3. EIC of a baby shampoo. The alkylamide column was used. The
mobile phase contained water with 50% ACN in the presence of 40 mM
HAcO/NaAcO buffer of pH 4.7. The flow rate was 0.2 mL min1. The inset
shows the m/z 399 trace with the intensity axis multiplied by 50.
Retention was much weaker with the cyanopropyl column; for instance,
using 20% ACN, the C8G1 isomers were eluted within 6 min (not shown). Using
95 and 100% water in the mobile phase, the C8G1 isomers appeared within 30
and 40 min, respectively. In all cases, the ring isomers were well resolved, but
the epimer pairs were poorly resolved with this column.
linearly varied from A to B in 20 min. The AMGs were separated with partial
resolution between the epimers, and with excellent resolution between the ring
isomers, within 12 min (not shown). However, the re-equilibration of this column
was observed to be rather slow. Pumping of mobile phase A during several hours
was required to achieve reproducibility between successive injections.
C12G1p
C16G1p
C8G1p
4
Abundance x 107
m/z 427
C16G1f
C10G1p
C12G1f
C14G1p
C14G1f
m/z 399
m/z 371
m/z 343
m/z 315
0
m/z 427
C10G1f
C8G1f
13 14
Time (min)
Time (min)
12
Fig. IV.4. EIC of Plantacare. The cyanopropyl column was used. The
composition of the ACN/water mobile phase (also containing 40 mM of
the HAcO/NaAcO buffer of pH 4.7) was linearly varied from 25 to 90%
acetonitrile in 15 min. The flow rate was 0.2 mL min1. The inset shows
the m/z 427 trace with the intensity axis multiplied by 20.
Using the cyanopropyl column, mobile phases containing 25:75 (A) and
90:10 (B) ACN/water (v/v) in presence of the acetic acid/sodium acetate buffer,
were used. The mobile phase was linearly varied from A to B in 15 min. As
shown in Fig. IV.4 for Plantacare, all the AMGs, from C8G1 up to C16G1, were
separated within 14 min. The epimer pairs were not resolved, but a good
resolution between the ring isomers was achieved. Successive injections of the
145
Abundance x 105
C12G1p
6
C14G1p
4
2
C14G1f
C12G1f
m/z 371
m/z 399
0
10
12
Time (min) 14
Fig. IV.5. EIC of a hand cream. The cyanopropyl column was used. The
composition of the ACN/water mobile phase (also containing 40 mM of
the HAcO/NaAcO buffer of pH 4.7) was linearly varied from 25 to 90%
ACN in 15 min. The flow rate was 0.2 mL min1.
146
concentration of the standards was varied from 10 to 120 g mL1 (from ca. 60
600 M for C1G1 to 30360 M for C12G1). In all cases, the calibration curves
gave a good linearity, with r2 > 0.99 (6 points per curve).
As shown in Fig. IV.6, the relative sensitivities of the -epimers (or
response factors, calculated by using -C1G1p as reference) increased almost
linearly from C1G1 to C8G1, reached a maximum at C10G1 and decreased for
C12G1. Relative rather than absolute sensitivities were plotted in this figure to
enhance the differences between the standards, independently from detector
sensitivity and some working conditions. A range of relative sensitivities for
C14G1 was also plotted on Fig. IV.6. This range was obtained by injecting
Plantacare, and using the sum of the areas of the C14G1p and C14G1f peaks, and
the composition given by the manufacturer (6.19.5% total C14G1 for a 5153%
pure product), to calculate the relative sensitivity range. As deduced from Fig.
IV.6, this range indicated a further decrease of the relative sensitivity for alkyl
chains longer than 12 carbon atoms. Since a range of concentrations was not
declared (Table IV.1), the relative sensitivity for C16G1 could not be calculated.
The influence of the mobile phase composition on the relative sensitivities
was also studied. For this purpose, a 20:80 (A) or a 75:25 (B) ACN/water (v/v)
mixtures, containing 20 mM NaAcO and 20 mM HAcO, were used. Dilutions of
the stock solutions of the standards were prepared using both A and B. The
dilution factor was 1:20, but 1:10 was used for C12G1. All the diluted solutions
also contained 50 g mL1 of the internal standard, -C9G1p. The autosampler
was directly connected with the ESI source with a PEEK tube in the absence of
the separation column, and the solutions were injected successively using A and
B as mobile phases. No significant differences between the peak areas obtained
with mobile phases A and B were observed. Thus, the differences among the
response factors observed in Fig. IV.6 should be attributed to the length of the
147
alkyl chains rather than to the increase of the acetonitrile concentration along the
elution gradient.
16
90
Relative sensitivity
50
8
LOD (M)
70
12
30
4
10
0
0
12
Fig. IV.6. Relative sensitivities (left axis and dashed line) of - () and alkylglycopyranosides () with respect to -C1G1, and LODs (, right
axis and dotted line) vs. the number of carbon atoms in the alkyl chain, n
(-C9G1p used as internal standard). All values were obtained from pure
standards, but for C14G1 the two points delimit the range of relative
sensitivity, which was estimated from its concentrations in Plantacare (6.1
9.5% C14G1 for a 5153% pure product, as declared by the manufacturer).
Taking into account that Na+ is bound to the same oxygen as the alkyl
chain
[Klaffke, 1999],
C10G1 can be due to stabilization of the adduct by the higher electronic density
provided by the longer alkyl chain. The smaller volatility, the formation of ionpairs or micellization are possible explanations for the sensitivity decrease when
the alkyl chain has 12 and 14 carbon atoms. Thus, the response factor can be
approximately predicted by linear interpolation within the C1G1C10G1 range,
but not when n > 10. Owing to the large differences, large systematic errors
148
149
0.30 for Plantacare and ca. 0.04 for the hand cream. Thus, the
furanoside/pyranoside concentration ratio can be used to characterize industrial
samples. However, owing to the lack of standards, only the total concentration of
each CnG1 group of isomers was obtained. For this purpose, the sum of the peak
areas of the pyranoside and furanoside forms, and the calibration curve of the
corresponding -pyranoside standard, were used. For Plantacare, the calibration
curves predicted concentrations within the ranges declared by the manufacturer
(Table IV.1). The analysis of a baby shampoo gave 0.5% C8G1, 0.2% C10G1,
and 0.6% C12G1. The peaks of the C14G1 isomers were also observed in the
chromatograms of this sample.
Table IV.1. Declared and found concentrations of alkylmonoglucosides in
Plantacarea
Compound Declared (%) Found (%)
C6G1
Max. 0.26
0.06
C8G1
12.2 15.9
13.0
C10G1
7.6 11.7
9.7
C12G1
18.9 22.3
18.9
C14G1
6.1 9.5
C16G1
Max. 2.12
[Harvey, 2000-A;
Harvey, 2000-B; Chiarelli, 1987; Cancilla, 1999; Harvey, 1994; Harvey, 1997; Kovik,
1995; Penn, 1996; Asam, 1997],
2005],
divalent cations
150
The
[Harvey, 2006]
Less attention has been paid to the CID spectra of monosaccharides and
their derivatives. The stereochemical differentiation of monosaccharides in the
presence of Zn2+
demonstrated.
[Gaucher, 1998]
The
positional
and
[Carlesso, 2000]
diastereomers
of
has been
sulfated
[March, 2005].
[Berman, 2006].
However, studies
151
[Kovik, 2001].
Using TOF-
[Hill,
[Rybinski, 1998;
aim of this work was to study the ion-trap stepwise fragmentation of AMGs,
which are the major components of industrial APGs. Also, the industrial
production of APGs leads to mixtures containing major amounts of AMGp, and
minor but significant concentrations of their five-membered ring isomers, AMGf.
Thus, in this section we describe the CID spectra of both AMGp and AMGf,
using several isotopic forms of D-glucose to assist interpretation. The successive
MS2, MS3 and pseudo-MS4 spectra of D-glucose, D-glucose-13C6, D-glucose-6,6d2, D-glucose-1-13C and AMGp having up to 12 carbon atoms in the alkyl chain
were obtained, at increasing fragmentation energies, with an ESI-IT-MS.
Standards of the corresponding AMGf were not available; however, previous
separation of a commercial mixture of APGs by HPLC, and their MS and MS2
spectra were obtained on-line.
152
octyl-
and
decylglucofuranosides
showing
much
lower
C6, both obtained at an energy of e = 0.45, are given. Using this fragmentation
energy, the peak of the parent ion was still observed together with the peaks of
next ion generation. By comparing the MS2 spectra of D-glucose and D-glucose13
C6, it is deduced that the m/z 185 peak of D-glucose corresponds to an ion with
six carbon atoms. This peak was interpreted as due to a loss of a water molecule
from the [M+Na]+ parent ion (m/z 203). At lower m/z values, the MS2 spectra of
D-glucose showed an abundant peak at m/z 143 and a weak one at m/z 113,
which according to MS2 spectrum of D-glucose-13C6 (Fig. IV.7, part B),
corresponded to ions with four and three carbon atoms, respectively.
153
1.5
413
[M+Na]+
Intensity x 103
143
1
0.5
593
185 203
571
557
113
0
100
200
400
500
m/z
600
147
413
[M+Na]+
Intensity x 103
1.5
300
0.5
191 209
116
0
599
100
577
563
200
300
400
500
m/z 600
Fig. IV.7. MS2 spectra of the [M+Na]+ parent ion of (A) D-glucose (m/z
203) and (B) D-glucose-13C6 (m/z 209), both obtained at e = 0.45 (, parent
ions).
0,2
A,
1,3
X,
2,4
X and
0,4
peak as parent ion, the MS2 spectrum of D-glucose-6,6-d2 (not shown) yielded a
peak at m/z 145. This indicated that carbon atom 6 was retained by the m/z 143
ion fragment of D-glucose. This excluded the
0,4
0,2
fragmentation. The two possible structures of this fragment ion are shown in Fig.
IV.8.
154
Similarly, the
0,3
A,
1,4
A,
0,3
X and
1,4
X fragmentations, plus a
3,5
Possible structures
OH
0,2A
Na+
5
m/z 143
OH
Na+
HO
OH
OH
OH
0,3A
m/z 113
O
Na+
HO
OH
6
Na+
OH
5
4
HO
Fig. IV.8. The two possible structures of the m/z 143 and m/z 113 ions of
the MS2 spectrum of the [M+Na]+ parent ion of D-glucose, produced by
0,2
Concerning to the abundant m/z 413 peak which was present in the MS2
spectrum of the [M+Na]+ parent ion of D-glucose, no shifts with respect to the
equivalent spectra obtained with either D-glucose-13C (Fig. IV.7, part B), Dglucose-6,6-d2 and D-glucose-1-13C (not shown) were observed. This indicated
the absence of fragments of glucose in the composition of the m/z 413 peak.
However, this abundant peak was also present in the MS2 spectra of all the
AMGs, and peaks showing the mass of the unfragmented molecules plus 413 m/z
155
units also appeared in the spectra of both D-glucose and all the AMGs. For these
reasons, further efforts to interpret the m/z 413 peak were performed. First, MS2
spectra of the [M+Na]+ parent ion of D-glucose were obtained both in the
absence of MeOH, and also using 20 mM NaHCO3 instead of 20 mM NaAcO. In
both cases, an m/z 413 ion, with a similar abundance as that observed using 50%
MeOH and 20 mM NaAcO, was obtained. Therefore, the presence of MeOH,
AcO- or HAcO in the composition of the m/z 413 ion was also discarded. On the
other hand, the m/z 413 peak was not observed when a 20 mM NaAcO solution
in the absence of D-glucose was infused. Thus, the m/z 413 peak was formed
only in the presence of D-glucose or an AMG, and could contain Na+, OH- and
H2O, but not carbon atoms.
Thus, fragmentation of the m/z 413 ion yielded peaks at m/z 301 and 189,
which corresponded to two successive losses of 112 Da, in the MS3 spectrum.
These losses could correspond to the uncharged fragment NaOH4H2O. A peak at
m/z 171 which was attributed to a loss of water with respect to the m/z 189 ion
was also observed. Further fragmentation of the m/z 301 ion was achieved by
setting the compound stability parameter at 300%. Again, the m/z 189 and 171
ions were exclusively observed on this pseudo-MS4 spectrum. Then, all the
possible combinations of Na+, OH- and H2O (up to 20 units of each, respectively)
matching m/z 413, with the restriction of having a single positive charge, were
computed. The following single combination was found: 4 Na+ + 3 OH- + 15
H2O. However, the exact mass of this combination, 413.1257 Da, did not match
with the m/z values of the peaks observed in the high-resolution spectra of Dglucose (413.2341 Da) and the AMGp (413.2349 - 413.2406 Da). Thus, no more
efforts to interpret the m/z 413 peak were done.
As deduced from the comparison of the MS2 spectra of Fig. IV.7, parts A
and B, the ion of D-glucose at m/z 593 had six carbon atoms. A likely
156
0,2
chain, this m/z value did not matched with the possible fragment ions obtained by
other cross-ring cleavages. This also agrees with the
0,2
A cross-ring cleavage
deduced above for D-glucose. The MS2 spectra of all the AMGp also showed an
m/z 129 ion which was not present in the MS2 spectra of D-glucose. This m/z 129
ion was weak for methyl-MGp and abundant for the other AMGp. This suggested
157
that the presence of the hydrocarbon chain was necessary to stabilize the
resulting non-ionic fragment. By computing all the possible combinations of C,
H, O and Na giving rise to an m/z 129 ion with a positive charge, the two
following possibilities were found: C4H10O3Na+ and C3H6O4Na+; however, a 2,5A
fragmentation is the only way to produce the former ion in a single step, whereas
the less likely concurrence of at least two fragmentations,
3,5
A
A
B1
[M+Na]+
Unknown
[M+413-2H2O]+
[M+413+H2O-NaOH]+
[M+413]+
Others
10
12
129
143
185
217
413
571
585
607
235
129
143
185
287
413
641
655
677
209
249
129
143
185
315
413
669
683
705
209
235
275
129
143
185
343
413
697
711
733
235
309
129
143
185
371
413
725
739
761
291
315
Concerning to the peaks at large m/z values, and as also occurred with Dglucose,
they
matched
with
the
following
compositions:
[M+413]+,
158
MGp, the abundant m/z 275 ion (Fig. IV.9) could be due to a loss of NaOH to
yield [M-OH]+. Finally, significant differences between the MS2 spectra of the and -epimer pairs of methyl- and octyl-MGp were not observed.
275
Intensity x 104
129
315
413
235
143 185209
0
100
200
669 683
300
400
500
600
m/z
705
700
octyl-MGp and octyl-MGf peaks, are shown in Fig. IV.11. At low fragmentation
energies (e < 0.6), the peak area of octyl-MGp was much higher than that of
octyl-MGf (see the MS-TIC chromatograms of Fig. IV.10, which correspond to
MS2-TIC with e = 0); however, at increasing energies, the peak area of the octylMGp decreased at a higher rate than that of the octyl-MGf.
octyl-MGp
Intensity x 108
1.5
1.0
decyl-MGp
0.5
octyl-MGf
decyl-MGf
m/z 315
0
m/z 343
2
5
Time (min)
As shown in Fig. IV.11, left part, the area of the octyl-MGp peak was even
smaller than that of the octyl-MGf peak when e = 0.80. Also, as observed in Fig.
IV.11, right part, the MS2 spectra of the alkyl-MGf differed ostensibly from
those of the corresponding alkyl-MGp. Thus, in comparison with the octyl-MGf,
the m/z 413 ion and its adducts with unfragmented molecules were more
abundantly formed by the octyl-MGp. The differences between the MS2-TICs
indicated an easier fragmentation of the alkyl-MGp compared to the alkyl-MGf.
160
150
100
50
octyl-MGf
octyl-MGp
e = 0.7
octyl-MGp
e = 0.7
315
80
octyl-MGf
octyl-MGp
Intensity x 104
413
octyl-MGp
e = 0.8
683
316
185 315
0.5
705
octyl-MGf
e = 0.8
185
669
octyl-MGp
e = 0.9
e = 0.9
octyl-MGf
octyl-MGp
143
413
315
413
e = 0.8
octyl-MGf
e = 0.7
143
20
315
413
705
octyl-MGf
e = 0.9
143
0.6
683 705
0.2
2
3
Time (min)
185
100
315
300
500
m/z
413
185
669
705
315
700
100
300
500
m/z
700
On the other hand, as also deduced from the MS2 spectra of Fig. IV.11,
the cross-ring cleavage at
0,2
the alkyl-GFs than in the alkyl-GPs. This could be due to a lower stability of the
five-membered furanoside rings in comparison to the six-membered pyranoside
rings. Finally, as also observed in Fig. IV.11, the alkyl-GPs and alkyl-GFs
showed a similar trend to lose the alkyl chain giving rise to the m/z 185 ion.
161
important surfactant classes, including alkyl ether sulfates and sulfonates, and
FAEs
[Farm, 2006.].
[Cheremisinoff, 2003].
but not FAEs with m >4, due to the limited volatility of these
compounds [Rudewicz, 1986; Crescenzi, 1995; Battersby, 2001]. The major drawback
of HPLC methods for FAEs has been the lack of an adequate detector [Rudewicz,
1986; Petrovic, 2001].
162
former, while the sensitivity of ELSD is poor for volatile compounds such as
non-ethoxylated alcohols (m = 0) and FAE oligomers with a low degree of
ethoxylation (m <3) [Miszkiewicz, 1996-A; Bernab-Zafn, 2006]. Finally, low LODs
are achieved with chromogenic and fluorogenic pre-column derivatization
procedures [Marcomini, 1996; Schmitt, 1990; Kiewiet, 1995; Lemr, 1994; Zanette, 1996;
Lemr, 1996; Sun, 1997; Hoffman, 2004-A; Lemr, 2003; Okada, 1991; Okada, 1992;
Hoffman, 2004-B; Bachus, 2003; Desbne, 2005; Heinig, 1996-A; Mic-Tormos, 2008-A;
Mic-Tormos, 2008-B; Mic-Tormos, 2009; Zu, 2010].
Chiron, 2000; Sherrard, 1994; Dunphy, 2001; Cassani, 2004; Sparham, 2005].
To
[Lemr, 1994;
Lemr, 1996; Lemr, 2003; Desbne, 2005; Heinig, 1996; Zu, 2010; Dunphy, 2001;
Cassani, 2004; Sparham, 2005].
oligomers with low m values by volatilization, the water content of the samples
must be reduced with particular care, which increases analysis time
2001].
[Dunphy,
163
[Holapek, 2005].
In this
164
but the medium contained also both 10% water and 40% ACN. These solutions
were infused in the MS, and the abundances of the [M-H]- ions were measured.
As shown in Fig. IV.12, sensitivity of the fatty acids increased as n
increased, and also varied largely with the nature of the infusion medium. Thus,
sensitivity increased ca. 5 times when butylamine was added to the extracts (Fig.
IV.12, from a to b). This was probably due to ionization of the acids in a
medium with a higher pH. Sensitivity further increased ca. 360 times when water
was also added (Fig. IV.12, from b to c).
1.2
C12E0A
C14E0A
Abundance x 107
C16E0A
0.8
C18E0A
100 x
0.4
0.0
a
Ii
I s [M ]i
(E.IV.1)
where Ii and Is are the abundances of the [M-H]- ion of the compound of interest
and the internal standard, respectively, and [M]i is the molar concentration of the
compound. As shown in Fig. IV.13, part A, relative sensitivities increased as the
alkyl chain increased. This agreed with the reported APCI-MS response factors
of fatty acids [Holapek, 2005]. Standards of ethoxy-carboxylic acids having large
values of both n and m are not commercially available; then, only the relative
166
log fi
m=3
m=2
m=1
-1
m=0
-2
-3
0
12
16
log fi
n=2
-1
n=1
-2
B
-3
0
Fig. IV.13. NI MS relative sensitivities of carboxylic and ethoxycarboxylic acids versus the number of carbon atoms in the alkyl chain, n,
and at increasing values of the number of EO units, m (A), and vice versa
(B). Relative sensitivities in logarithmic scales, log fi, are given with
reference to the internal standard (TMBA).
167
[Miller,
was 1.3% of the slope, which corresponded to a confidence limit of 3.0% (95%
confidence
level,
two-sided).
Using
increasing
concentrations
of
the
168
addition with a syringe pump under stirring during 40 min. The abundance of the
[M-H]- ions, measured in the NI mode, was compared to that obtained using
calibration curve B (expected concentration in the infused solutions, ca. 200 g
mL-1). Either, dropwise or slow addition of the Jones reagent equally led to a
100% yield.
The influence of the reaction temperature during oxidation was also
investigated. Alcohols with a short (C3E0) and a large (C12E0) chain length
were used. A water bath was used to set the initial temperature of the reaction
mixture. Then, the Jones reagent was added, and after extraction the abundance
of the [M-H]- ions was measured. The ion abundances did not increase nor
decrease by regulating the initial temperature of the reaction mixture to 25, 35
and 55 C.
diluted using acetone, and the proposed procedure was applied; however,
oxidation was performed with the series of Jones reagent prepared with
increasing acetic acid concentrations (decreasing water concentrations). Second,
aliquots of the C10E0 stock solution were diluted with acetone-water mixtures
containing increasing amounts of water, instead of using pure acetone. Jones
reagent prepared in aqueous sulfuric acid, as indicated in the proposed procedure,
169
was added to these solutions. In all cases, the NI spectra were obtained, and
internal standard correction using TMBA was applied.
As shown in Fig. IV.14, the yield was maximal when the reagent was
prepared as indicated in the proposed procedure, and decreased moderately when
Oxidation yield, %
water was partially substituted by acetic acid (Fig. IV.14, part A).
100
80
60
Oxidation yield, %
40
20
40
60
80
H2O % in the Jones reagent
100
80
60
40
0
25
50
75
100
Initial H2O % in the sample solution
Oxidation yield also decreased when the Jones reagent was added to C10E0
solutions containing increasing amounts of water (Fig. IV.14, part B).
Therefore, in the recommended procedure, industrial samples containing water
(i.e. cosmetics and body care products) were diluted with acetone. In addition,
170
171
peaks corresponding to the C12E3 - C12E3A pair were observed in the spectrum
obtained after oxidation of C12E3. The peak of the remaining non-oxidized
C12E3 was higher than that of C12E3A; however, taking into account that the
C12E3/C12E3A sensitivity ratio in the PI mode was close to 1.8, an oxidation
yield of ca. 60% was calculated. Two other low intensity peaks, corresponding to
the [M+H]+ ions of the C12E2 - C12E2A pair, were also observed. These two
peaks were attributed to loss of an EO unit during oxidation. From the peak
abundances, this side-reaction amounted to less than 4% of the initial C12E3
concentration. Along the C12Em series, oxidation yield, which was 100% for
C12E0, decreased to ca. 65 and 60% when m = 2 and 4, respectively. The
oligomers with m = 5 and 6 also gave a ca. 60% yield.
[C12E3+H]+
[C12E3A+H]+
Abundance x 106
1
[C12E2+H]+
[C12E2A+H]+
0
300
m/z
172
350
Finally, ethoxylated alcohols of the CnE1 and CnE2 series were used to
study the influence of n from n = 2 to 18. In all cases, ca. 65% yields were
obtained. In addition to the expected peaks, two additional peaks due to the FAE
with loss of an EO unit, and its corresponding ethoxy-carboxylic acid, were
observed. However, yields for this side reaction were small (4-8% of the initial
amount of the FAE).
173
500
m/z
[C10E13A-H]-
[C10E12A-H]-
[C10E11A-H]-
[C10E9A-H]-
[C10E8A-H]-
[C10E7A-H]-
[C10E6A-H]-
[C10E5A-H]-
[C10E2A-H]-
[C10E1A-H]IS
300
[C10E10A-H]-
[C10E0A-H]-
Abundance x 106
[C10E4A-H]-
[C10E3A-H]-
700
174
Table IV.3. Solutions of C16E0A and C18E0A were used to construct external
calibration curves (6 duplicated points). To check possible matrix effects,
internal calibration by the standard addition method was also applied; for this
purpose, two duplicated calibration points per sample were performed (with
addition of alcohol standards to the samples before the oxidation step).
[C18E0H-H]-
Abundance x 106
[C16E0H-H]4
IS
0
200
250
m/z
300
175
in the MS, LODs of 25 and 7.5 g per gram of sample, respectively, were
calculated.
Table IV.3. Mass percentages of cetyl and stearic alcohols found in several
commercial samples by external and internal (standard addition) calibration (ND =
not detected).
C16E0 (%)
Sample
Shampoo
Hair conditioner
Lip protector stick
Deodorant stick
Body-care oil
Varicose-vein cream
External
0.71
0.55
0.66
ND
0.10
3.8
Internal
0.80
0.35
0.69
ND
0.16
3.3
C18E0 (%)
External
ND
0.25
0.34
5.0
0.15
2.0
Internal
ND
0.24
0.34
5.6
0.15
2.6
C) Seawater
The presence of saturated and unsaturated fatty alcohols at the g L-1 level
has been reported in seawater samples taken from coastal and high seas
1992; Tolosa, 2003; Belanger, 2009].
[Parrish,
correspond to linear fatty alcohols with zero, one and two double bonds, and to
phytol (a common diterpenoid), were observed in the spectra of the oxidized
extracts obtained with seawater. These peaks were present with much lower
abundances in the spectrum of the seawater extract which was not oxidized with
the Jones reagent. In Fig. IV.18, ratios calculated by dividing the ion
abundances obtained after application of the proposed procedure to the seawater
extract, by those obtained without oxidation with the Jones reagent (reference
sample), are indicated between parentheses.
176
C26:1 (12)
C26:2 (30)
C24:2 (10)
C22:0 (2)
C22:2 (25)
C22:1 (4)
C20:2 (92)
C20:1 + phytol (13)
C20:0 (4)
C18:1 (10)
C18:0 (15)
C16:1 (16)
C16:0 (30)
0.5
C14:0 (8)
1.0
C12:1 (140)
C12:0 (13)
Abundance x 106
C12:2 (600)
1.5
0
200
300
m/z
177
400
CHAPTER V
ANIONIC SURFACTANTS
V.1. AESs
[Fiedler, 1989].
2006; Belanger, 2006; Van Compernolle, 2006; Ribosa, 2007; Jurado, 2007].
Both the
181
[Rudewicz, 1986;
for underivatized FAE oligomers decrease ca. two orders of magnitude when m
decreases from 4 to 1
2001].
[Trathnigg,
or CE
Dunphy, 2001; Lemr, 2003; Desbne, 2005; Wallingford, 1996; Heinig, 1998],
have
been also described. FAE derivatives have been separated by either NP- or RP-
182
or MS detection
[Crescenzi, 1995; Bernab-Zafn, 2006; Levine, 2005; Jandera, 1998; Krogh, 2002].
[Llenado, 1983].
and
[Smedes, 1982-B].
[Neubecker, 1985],
as well as
have
been applied to the determination of AES in waters, sewage sludge and marine
sediments.
In former works of research group, we have developed procedures for the
RP-HPLC-UV determination of FAE previous derivatization with a cyclic
anhydride
Tormos, 2010].
AES to yield exactly the same derivatives as FAE, i.e. the hemiesters of the
alkyl- or alkyl-ethoxy residues. Thus, the peaks corresponding to the sum of both
FAE and AES oligomers are obtained on the chromatograms if the two surfactant
classes are present in the samples. Therefore, in this work, a procedure for the
separation of these two surfactant classes, followed by the independent
derivatization of each class with a cyclic aromatic anhydride, and RP-HPLC-UV
determination of the derivatized oligomers, was developed. Separation of the two
surfactant classes was achieved by SPE on a SAX cartridge. Then, using either
phthalic or diphenic anhydride, FAE are esterified and AES are transesterified.
183
Thus, to assure
100
Formation of
diphenates
80
60
Formation of
phthalates
40
20
0
0
40
80
Reaction time (min)
Fig. V.1. Relative sum of the chromatographic peak areas of the derivatives
of all the oligomers given by an industrial AES (LES) sample versus
reaction time using phthalic (rhombus and dashed line) and diphenic
anhydrides (squares and dotted line). Each point represents an independent
derivatization performed at 105 C in 1,4-dioxane.
184
300
200
C12E0 C12E1
+
+
C12E5 C12E4
C12E6
C12E7
C12E8
C12E3
C12E9
C12E2
n = 12
n = 14
n = 16
n = 18
100
0
n = 12
300
200
100
C12E0S
+
C12E5S
C12E1S
+
C12E6S C12E4S
C12E3S
C12E7S
C12E8S
C12E2S
C12E9S
n = 14
n = 13
n = 15 n = 16
n = 18
0
10
20
30
Time (min)
185
40
Except for m = 0 and 1, the oligomers within the series eluted by following the
order of decreasing m. The consecutive pairs of oligomers were also fairly well
resolved; however, the peaks of the oligomers with m = 1 and 0 overlapped with
other oligomers within their respective hydrocarbon series. At 25 C and for the n
= 12 and 14 series, overlapping of the pairs m = 1 and 4, and m = 0 and 5, was
produced. Reversion of the elution order for m = 1 and 0 within the hydrocarbon
series has been explained as due to the rigidity of the short hydrophilic moiety of
these oligomers of the EO chain, which hinders intramolecular solvation, thus
making their hydrophobicity to decrease with respect to the oligomers with m 2
[Mic-Tormos, 2008-A; Mic-Tormos, 2008-B; Mic-Tormos, 2009; Mic-Tormos, 2010].
[Fendinger, 1992].
186
the cartridge with additional portions of 50% MeOH. In this way, residual
cations from the sample (mostly, Na+) were washed away, thus strongly fixing
AES on the SAX cartridge before increasing the hydrophobicity of the medium.
Experiments performed with LES in 50% MeOH showed the absence of AES
oligomers in the chromatograms obtained by increasing MeOH concentration to
80% after washing the cartridge first with more 50% MeOH.
300
200
100
40
20
0
10
20
30
Time (min)
40
188
in Fig. V.2, part A, whereas the chromatogram obtained with the combined
fractions 4 + 5 showed no significant peaks. Therefore, FAE were quantitatively
retained in the combined fractions 1 + 2 + 3.
A
Absorbance at 230 nm (mAU)
100
50
B
20
0
20
30
Time (min)
40
Similarly, using LES, the chromatogram obtained for the combined fractions 4 +
5 was closely similar to that observed in Fig. V.2, part B. On the other hand, the
chromatogram obtained with the combined fractions 1 + 2 + 3 showed a series of
small peaks which followed the same pattern as that observed in Fig. V.2, part
B, but with much smaller peak areas. This later chromatogram was attributed to
the FAE impurities which are always present in industrial AES, due to the nonquantitative sulfatation of FAE during AES manufacture. The total peak area
189
obtained from fractions 4 + 5 of LES, divided by the total peak area obtained
from fractions 1 + 2 + 3 indicated a molar percentage of ca. 1.2 % FAE in the
LES sample.
[Mic-Tormos, 2009],
[Mic-Tormos, 2008-B]
and diphenic
190
In mass percentages; the average EO number, m number found for each AES
sample is indicated between parentheses.
[Mic-Tormos, 2008-B].
As
shown in Fig. V.5, the peak areas of the m = 5 and 4 oligomers were estimated
by non-linear interpolation of the peak areas of the 2 m 3 and m 6
oligomers of their respective hydrocarbon series. Thus, the peak areas of the
191
Dehydol LT-7 ( m = 7) and LES ( m = 3) fitted well to the cubic equation, and to
exponential equations of the form A=be-am, respectively.
FAE, m = 7
n = 12
Relative area
AES, m = 3
n = 12
n = 14
0
2
n = 14
10
12
Number of EOs
Fig. V.5. Estimation of the uncorrected peak areas of the m = 4 and 5
oligomers of the n = 12 and 14 series in: Dehydol LT-7 (empty symbols,
solid lines by cubic interpolation); LES (full symbols, dashed lines by
exponential interpolation). Experimental (rhombus) and interpolated peak
areas (squares).
For simplicity, when the peak areas of the m = 4 and 5 oligomers of the n = 12
and 14 series of Dehydol LT-7 were estimated, the contribution of the oligomers
with m > 13 of the n = 14 and 16 series (see Fig. V.2), respectively, was
neglected. Then, the peak areas of the m = 0 and 1 oligomers were obtained by
difference. All the peak areas were next divided by the tabulated response factors
of the respective oligomers
and the
193
process and the quality of the final product. As indicated in Table V.1,
application of the proposed procedure to the 70% generic LES sample showed a
65% AES and a residual 1.2% FAE. According to the data given in Table V.1,
the proposed procedure is also useful for the characterization and determination
of both FAE and AES in commercial cleaners.
Absorbance (mAU)
40
FAE
n = 12
AES
n = 12
20
20
0
0
Intensity 105
AES
n = 14
2
0
2
3
24
26
24
22
Time (min)
2
1
0
22
26
0
30
Time (min)
32
34
Time (min)
Using both UV-Vis and MS detection, the seawater samples showed measurable
amounts of several FAE oligomers of the n = 12 series, and several AES
194
195
CHAPTER VI
SYNTHETIC POLYMERS
VI.1. PVP-NO
DTIs, act as dye scavengers, keeping the washed-off dyestuffs in solution, thus
helping in the prevention of redeposition on the fabrics. For years, PVP and their
copolymers have been mainly used; however, the dye scavenger efficiency of
these polymers is reduced in the presence of anionic surfactants [Oakes, 2003]. For
this reason, new generations of DTI, with superior complexing properties and a
higher tolerance to anionic surfactants, have begun to be employed. A relatively
common DTI of the new generation is PVP-NO (see structure in Fig.
VI.1).Molecular masses between 9 and 36 kDa, which correspond to polymer
chains constituted by ca. 75 and 300 monomers, respectively, are found in
different types of commercial PVP-NO for laundry
199
[Gallardo, 1999].
and MEKC to the analysis of synthetic polymers has been reviewed [Cottet, 2005].
Both FSCE and MEKC can provide useful information about the behavior of
polymers in solution, as well as quantitative information
MEKC, Gallardo et al.
[Gallardo, 1999]
[Bohrisch, 2000].Using
[Aguilar, 2002].
[Grosche, 2000],
[Bohrisch,
2000;
a cellulose
Clos,
1998;
[Bohrisch,
actually give two or more signals at different migration times rather than a single
peak.
200
[Cottet,
Using FSCE, two peaks, which were attributed to the very slow rate of
exchange between the free and aggregated or micellized states of the polymer in
comparison to the time scale of electrophoresis, were observed. The free chains
of the polymer (the unimers) exhibited a larger mobility (in absolute values) than
the micellized polymer. Further, the peak of the micellized polymer was broader
than that of the free polymer, which was attributed to scatter in the aggregation
numbers.
Using FSCE and a PVP-NO/ maleic acid anionic copolymer, Gyrffy et
al.
[Gyrffy, 1998]
presence of two components in the polymer was proposed, but the possible
nature of the components was no further discussed. Using both SEC and FSCE,
tpnek et al.
[tpnek, 2001]
in solution, and that the micelles coexist in a mobile equilibrium with the
unimers. Further, the unimer-micelle equilibrium was shown to be kinetically
frozen in aqueous media, the micelles behaving as independent nanoparticles.
In this work, a narrow peak was obtained for PVP-NO solutions using
both FSCE and MEKC; however, at least an additional broad band was also
present in most electropherograms. The nature of the signals obtained is
discussed at the sight of both the studies reported in the literature and the new
results. In addition, low-molecular-mass zwitterions, including trimethylglycine
(betaine) and sarcosine, have been used for a long time to reduce adsorption in
the FSCE separation of proteins
[Bushey, 1989];
have carboxylate groups, which hinders their application below pH 56. Thus, to
separate proteins in more acidic media (pH 25), PEA has been used [Chen, 1992].
201
Abs. (mAU)
showed the spectrum of the polymer (see the insets in Fig. VI.1).
CH2 - CH
40
N+
O-
40
20
0
8
EOF
20
0
200
300
(nm)
400
0
0
8
Time (min)
10
Fig. VI.1. Electropherograms of an EOF marker (mesityl oxide) and PVPNO (2000 g/mL) obtained using positive polarity and a BGE containing
10 mM HAcO (pH 4). The insets show the chemical structure of PVP-NO,
and the UV spectra at both the peak maximum and at the baseline location
indicated by an arrow.
Taking into account the large molecular masses of this polymer (917 kDa), and
its nominally nonionic nature, a single peak at the EOF migration time was
actually expected. However, as discussed below in Section VI.1.1.2, the
202
[Hilal, 1995]
ionization of PEA: 1.07, 5.75 and 8.91; thus, the PEA species without a net
charge predominates, at least on a 9:1 basis, within the 2.074.75 pH range.
Therefore, a BGE containing 20 mM H3PO4 (pH 2.2) was used to investigate the
effect of the PEA concentration on the migration behavior of PVP-NO and the
EOF. As shown in Fig. VI.2, using this BGE in the presence of increasing PEA
concentrations, PVP-NO gave a large narrow peak (after an initial small peak)
within the anionic migration region. A broader band at the beginning of the
cationic migration region (immediately after the peak of the EOF marker) was
also observed. Both the large narrow peak and the band exhibited the spectrum of
the polymer. Except when otherwise indicated, adsorption along the baseline
after the peak locations was not detected by using 150 mM or higher PEA
concentrations.
Contrary to what occurred in Fig. VI.1, negative polarity was required in
Fig. VI.2 to observe the PVP-NO peaks. As revealed by using acetone as a
marker, the EOF was reversed in the presence of large PEA concentrations. The
negative EOF could be due to the formation of esters between the phosphate
groups of PEA and the silanols. This would lead to the predominance of the
203
positively charged amino groups of PEA covering the capillary walls, thus
A
40
B
20
0
0
8
12
Time (min)
204
at
increasing
capillary
temperatures,
from
25
to
45C
205
peak varied from -17.6 to -18.2 and -19.7 (in 109 m2s1V1) when the capillary
temperature was increased from 25 to 35 and 45C, respectively.
200
160
B
120
C
80
D
40
E
0
0
12
16
Time (min)
206
supported by other reports from literature, the anionic character of PVP-NO can
be explained by the separation of charges along the N-O bond, and the
subsequent formation of a ionizable complex with water. A possible explanation
for the additional broad band is the presence of a micellized form of the polymer.
The nitrogen and oxygen atoms of pyridinium oxides exhibit a remarkable
separation of the respective positive and negative charges
[Ochiai, 1953].
This
[Okamoto, 1998].
presence of strong interparticle forces, which result from the charge separation
across the N-O bond
[Lee, 1996].
[Yamamoto, 1996].
[Yamamoto, 1996].
Finally, purified
PVP-NO fractions gave solutions with pHs as low as 3.44.5 when solved in
water, which was attributed to the formation and subsequent ionization of a PVPNO/water complex [Okamoto, 1998].
The formation and subsequent ionization of a PVP-NO/water complex
fully agrees with the net anionic electrophoretic mobility of the large narrow
PVP-NO peak observed in this work. Further, the acidity of some N-O bonds
along the polymer chain can be enhanced by the presence of neighboring N-O
bonds, as occurs with polycarboxylic acids [Gyrffy, 1998], which would lead to a
range of pKa values along the polymer. This would also explain the increase of
207
the net anionic mobility showed by this PVP-NO peak (in absolute terms) at
rising pH values from 2.2 to 3.0 (Figs. VI.2, part B and VI.3).
Another point to be explained is the presence of the broad bands near the
EOF migration time. At the sight of the previous studies
1998; tpnek, 2001],
of the polymer. These associated or aggregated forms could coexist with the free
form (the unimers) in a very slow or frozen equilibrium between them, as
reported for some copolymers
[tpnek, 2001].
208
problem was not overcome by conditioning the capillary between injections with
100
50
0
0
Time (min)
Lack of reproducibility was attributed to adsorption, then, the behavior of PVPNO at increasing SDS concentrations was studied in the presence of 250 mM
PEA. Under these conditions, and using pH 3.0 (20 mM NaH2PO4), PVP-NO
showed the reproducible but complex behavior which is outlined in Fig. VI.5.
Between 1 and 3 mM SDS (Figs. VI.5, parts A and B), a single peak at
increasingly longer migration times was observed. With 3 mM SDS, a new broad
band at a short migration time, and a new narrow peak, began to appear. The
band increased with 5 mM SDS, and reached a constant intensity within the 20
60 mM SDS range (Figs. VI.5, parts C-F). The narrow peak was large at 520
mM SDS (Figs. VI.5, parts C and D), and appeared at longer migration times
and became broader at higher SDS concentrations (Figs. VI.5, parts E and F).
Using 20 mM SDS and 35C (instead of 25C), an electropherogram similar to
that shown in Fig. VI.5, part D was obtained, but with the large narrow peak
located at a longer migration time (ca. 10 min). A possible explanation of the
results summarized in Fig. VI.5 is next given and discussed.
209
200
C
100
D
E
F
0
0
10
Time (min)
20
30
SDS. Mixed PEG-SDS micelles have a definite composition, with the excess
material, whether polymer or SDS, being present as free polymer molecules or
regular SDS micelles, respectively. When SDS is added to a solution of the
polymer, two concentrations, x1 and x2, mark the beginning and the completion of
the association of SDS with PEG, respectively. The association process starts
abruptly above a certain surfactant concentration, x1, which is lower than the
CMC, and saturates abruptly above another surfactant concentration, x2, where
210
the coverage of the polymer by adsorption of surfactant molecules along its chain
is completed up. Beyond x2 the surface tension of the solution is close to that of a
solution containing regular surfactant micelles. The difference, x2 - x1, measures
the amount of surfactant bound to the polymer.
According to NMR studies
[Cabane, 1977],
[Arai, 1971]
tension vs. SDS concentration curves when recorded in the presence of PVP.
Association of SDS with PVP to form mixed micelles began at an SDS
concentration 40% lower than the CMC. The PVP/SDS weight ratio at the point
where adsorption was completed, x2, was 1:2.3, regardless of the PVP
concentration. This corresponded to 1.1 PVP monomers for one SDS molecule.
According to literature, PVP-NO also interacts strongly with the SDS
micelles. Thus, the inhibition of the dye scavenger capability of PVP-NO in the
presence of SDS and other anionic surfactants has been attributed to
displacement of the dye from the polymer/dye complex to form a
polymer/surfactant complex
[Gyrffy, 1998]
[Oakes, 2003-A].
211
separated from the hydrophilic regions, the interaction was explained by the
formation of mixed polymer/SDS micelles, in which short segments of the
polymer occupied substantial parts of the micelles. As illustrated in Fig. VI.6, in
these parts of the micelles, a segment of the hydrocarbon skeleton was proposed
to be located inside the micelle, and the polar pyridine N-oxide groups were
assumed to be positioned among the SDS sulfate groups. Since a single polymer
molecule can bind several micelles, mixed aggregates containing a number of
SDS micelles was proposed to be formed
C].
OO-
N+
SO2
[Oakes, 2003-C],
Curves of the surface tension against the SDS concentration, obtained both
in the absence and in presence of 1000 g mL-1 PVP-NO, are shown in Fig. VI.7.
These measurements were made in the conditions of Fig. VI.5, that is, in the
presence of both 20 mM NaH2PO4 and 250 mM PEA. In the absence of the
polymer, the CMC of SDS was 1.8 mM. This parameter, which is 8.3 mM in
pure water, decreases in the presence of salts
which is consistent with the value obtained. Two transition points, indicating the
212
beginning and end of the association process of PVP-NO with the SDS micelles,
were observed in the presence of 1000 g mL PVP-NO.
70
60
50
40
30
0
0.5
1.5
log ([SDS]+1)
The first point, x1, was located at 0.48 mM SDS, which corresponds to a
concentration a ca. 70% lower than the CMC. This indicates a strong interaction
between the polymer and SDS. The second point, x2, was placed over the CMC,
at 9.7 mM SDS. The difference (x2-x1) corresponded to 0.9 PVP-NO monomers
for one SDS molecule taking part in the mixed micelles. This value is close to
that reported for the formation of PVP/SDS mixed micelles, 1.1
[Arai, 1971].
Assuming that PVP-NO would behave in a similar way as PEG, a mixed micelle
would have about 70 SDS molecules with only a 10% of the polymer taking part
of the micelles
[Cabane, 1977].
SDS molecule would correspond to 6.3 PVP-NO monomers per mixed micelle,
which is quite realistic. This value was used to draw the scheme of Fig. VI.6.
213
[tpnek, 2001],
polymer, namely, the free polymer and the pure polymer aggregates, probably in
a very slow-rate equilibrium between them, could be present in the aqueous
solutions of PVP-NO, in the absence of SDS. Further, in agreement with the
study of Oakes et al.
[Oakes, 2003-C],
forms could bind SDS micelles. Thus, it seems reasonably to assign the narrower
peaks of the electropherograms of Figs. VI.4, part A, VI.5, part C and D to the
association between the free polymer and SDS micelles, and the broader band at
a shorter migration time to the association between the aggregated polymer and
SDS micelles. In this latter, band broadening could be explained by scatter of the
aggregation number. The presence of two types of PVP-NO/SDS aggregates
could explain the persistent presence of a peak and a band (or group of bands) in
most electropherograms, being also compatible with the presence of the two
transition points observed in Figs. VI.7.
The EOF should be almost zero at the low pH used; however, as indicated
above, an anionic (reversed) EOF was observed in the presence of large PEA
concentrations. Thus, the reduction of this cathodic EOF produced by the
presence of increasing SDS concentrations could be enough to explain the
broadening and shifting effects on the large narrow peak observed in Fig. VI.5,
parts A and B. The first transition point of Fig. VI.7, x1, approximately
coincided with the appearing of the new peak and band between Fig. VI.5, parts
A and B. Thus, the sudden formation of two new peaks between 3 and 5 mM
SDS could be attributed to the simultaneous formation of both free polymer/SDS
and aggregated polymer/SDS mixed micelles.
Since the EOF was very low in the conditions of Fig. VI.5, parts B to F,
the short migration times of the band and peak indicated that the polymer species
214
had high anionic mobilities. These can be explained by both the negative charge
of the free polymer, and the association of the free and aggregated polymer forms
with the SDS micelles. As shown in Fig. VI.5, parts E to F, the band assigned to
the associated PVP-NO/SDS mixed micelles showed no further changes of shape
and location at higher SDS concentrations. On the contrary, the peak assigned to
the free polymer/SDS mixed micelles was progressively broader, and appeared at
longer migration times as the SDS concentration increased. An explanation for
this behavior was not found.
commercial
DTI
concentrates
for
laundry.
representative
215
According to the calibration curve, the area of the narrow PVP-NO peak
corresponded to 161 g mL-1 in the injected solution, and to 1.53% in the sample.
Table VI.1. Migration time and peak area repeatabilities, efficiency and LOD for
the large narrow peak of PVP-NO.
Parameter
Value
0.30a), 1.0b)
2.4a), 5.5b)
N, m1
80 000
23
a)
b)
20
12
16
20
Time (min)
216
VI.2. PVP
purposes,
including
cosmetics
and
toiletries,
[Frauenfelder,
1974;
textiles
Sheth,
and
1985].
dyes,
In the
[Behen, 1964].
[Dwyer, 1964],
and by
217
[Chmilenko, 2001-A].
[Jones, 2004];
Barkin, 1955; Frank, 1957; Luck, 1958; Molineux, 1961; Runge, 1996; Chmilenko, 2001B; Oakes, 2003-C].
PVP
C H C H2
N
O
n
NH2
O
N
O
S
O
Na+ O-
O- Na+
S
O S
O- Na+
O
H2N
NH
O
Na+ O-
Fig. VI.9. Molecular structures of PVP and the azo-dyes used in this work.
219
A
60
Absorbance (mAU)
6
40
4.5
3.0
F 2.25
20
G 1.12
0
0
10
Time (min)
Fig. VI.10. Electropherogram of 0.5 mM CR (A) and electropherograms of
500 g mL1 PVP of 60 kDa (4.5 mM in monomers) without an azo-dye
(B), and in the presence of the following CR concentrations: 0.75 (C), 1
(D), 1.5 (E) and 2 mM (F). Trace G was obtained with 4 mM AB. The
numbers on the traces are the values of q (monomer/dye molar ratio). The
traces were recorded at 215 (B), 500 (A, CF) and 565 nm (G). On each
trace, the arrow indicates the location of the EOF marker peak. The area at
the left half of the peak of the PVPdye complexes, a, was used to estimate
the total area of this band (a = APVPD/2).
A significant parameter in all the experiments along this work was the
monomer/dye molar ratio, which will be indicated by q throughout the article. At
increasing CR concentrations (decreasing values of q), the band appeared at
increasing migration time values after the EOF time. The band was also
220
progressively divided into two peaks which showed a large absorptivity in the
visible region, with their respective maxima at ca. 505 and 485 nm, respectively.
At q < 4, the peak with a lower migration time was Gaussian shaped, whereas the
other peak was asymmetric (Fig. VI.10, parts E to F). Resolution between the
two peaks, and their areas at 500 nm, increased as q decreased; however, the area
of the Gaussian shaped peak remained constant when q < 4. The asymmetric
peak continued increasing, approaching the migration time of CR at q <4 (Fig.
VI.10, parts E and F). Interpretation of the electropherograms was made in the
light of both the UVVis spectra of the free dye and the PVPCR complexes,
which were obtained with a conventional spectrophotometer, and the NECEEM
theory (see Section VI.2.1.3).
Using a conventional spectrophotometer, the absorption spectrum of CR
showed two maxima located at 340 and 487nm ( = 16,845 and 21,400 M1 cm1,
respectively). Upon addition of an excess PVP (0.91 mM in monomer giving q =
9.1), the band at 340 nm was only slightly modified, but the maximum of the
other band shifted from 487 to 505 nm. The molar absorptivity of this band also
increased from 21,400 to 24,000 M1 cm1. Batochromic shifts between 7 and 19
nm have been reported for the 500650 nm band of azo-dyes upon complexation
with PVP
in this work for the formation of the PVPCR complexes. Accordingly, the
Gaussian shaped peak of the electropherograms of PVPdye mixtures (Fig.
VI.10, parts B to F), was attributed to the corresponding PVPdye complexes,
and the asymmetric peak at a longer migration time to the free dye. As also
shown in Fig. VI.10, when q < 4, the asymmetric peak was close to the location
of the free dye peak obtained in the absence of the polymer (trace A). There are a
large number of potential binding sites along a PVP molecule. Thus, the increase
in the peak area of the complexes at rising dye concentrations was attributed to
221
an increased number of bound sites along the PVP molecules. The increasing
mobility of the complexes at increasing dye concentrations (at decreasing q
values up to q = 4) was also attributed to the same cause.
On the other hand, at q > 4 (Fig. VI.10, parts C and D), the peak of the
free dye ions was located at a migration time which was only slightly higher than
that of the PVPCR complexes. Further, a similar behaviour was observed by
injecting PVPAB mixtures. The peak due to the free dye was expected at longer
migration times, namely, at 6.4 and 9.5 min for CR and AB, respectively;
however, a peak close to these locations was obtained only when q < 4. As
further discussed in Section VI.2.1.3, this, as well as the presence of an
intermediate exponential region when q < 4, were explained by applying
NECEEM concepts.
222
when a targetprobe
223
to their respective electrophoretic mobilities. Since both target and probe are
carried away from the complex during migration, the complex dissociation rate
depends exclusively on its concentration. For this reason, a first-order kinetics,
which gives rise to an exponential liberation of both target and probe, is
followed. The stability constant of the targetprobe complex can be established
by measuring the peak area of the free probe, and the sum of the peak areas of
the remaining complex and the exponential region. Correction of the areas is
necessary if the complex and the probe have different sensitivities (e.g. different
fluorescence quantum yields or different molar absortivities). Finally, kinetic
information related to the complex dissociation rate can be obtained from the
exponential profile of the intermediate region.
The PVPdye mixtures used in this work gave rise to electropherograms
which closely resembled to those described for proteinprobe and DNAprotein
complexes when injected in NECEEM conditions. As shown in Fig. VI.10, parts
C to G, the Gaussian peak attributed to the PVPdye complexes was followed by
an exponential region which ended abruptly at the long migration time side;
however, a peak due to the equilibrium concentration of free dye could not be
distinguished in the electropherograms of Fig. VI.10. This was attributed to the
use of q values not sufficiently close to the maximal stoichiometry of the
complexes, which as shown later in Section VI.2.1.5, was q = 4. In fact, in Fig.
VI.10, this ratio was 4.5 and 3.0 for traces D and E, respectively. At large q
values, the excess PVP should reduce the equilibrium concentration of the free
dye below its detection limit. On the other hand, at too low values of q, the large
equilibrium dye concentration could not be distinguished from the much lower
amount of dye liberated by the complexes. However, as shown in Fig. VI.11, a
peak of the free dye, separated from the beginning of the exponential region, was
224
Absorbance (mAU)
A
6
20
3.1
Absorbance (mAU)
Ae
4.0
6
7
Time (min)
3
40
AD
3.1
2
Ae
4.0
5
6
Time (min)
Absorbance (mAU)
AD
C
80
3.1
10
AD
Ae
4.0
2
7.0
7.5
Time (min)
Time (min)
Fig. VI.11. Electropherograms of PVP of 10 (A), 60 (B) and 360 kDa (C)
with 4 mM CR at the values of q (monomer/dye molar ratios) indicated on
the traces. The insets show how the areas corresponding to AD and Ae were
established. The electropherograms were recorded at 500 nm. Other details
as in Fig. VI.10.
due to the equilibrium concentration of the free dye, AD; and (iii) the area due to
the dye liberated from the complexes during separation, Ae. The values of these
three areas were measured as next explained. As indicated in Fig. VI.10, the
maximum of the PVPdye peak was first located, and the area of the left half of
this peak was taken as APVPD/2. Then, as indicated in Fig. VI.11 for the
experiments performed at 3.1 q 4.4, the area at the right of the small dip of
the asymmetric peak was assigned to the equilibrium concentration of the free
dye, AD. Finally, the area of the exponential region, Ae, was calculated as the total
area minus (APVPD + AD). These areas were used below in sections VI.2.1.5 and
VI.2.1.7 to estimate the maximal stoichiometry and average stability constant of
the PVPdye complexes, respectively.
VI.2.1.4. Influence of MW on the location and shape of the peak of the PVP
dye complexes
As deduced from the electropherograms of Figs. VI.10 and VI.11, when q
< 4 (the dye was in excess in relation to the saturation point), the mobility of the
PVPdye complexes was independent of the molecular mass of the polymer.
This agreed with the model of repeating units of polymer binding individual ions.
A single peak, at a migration time independent from the polymer molecular mass
should be obtained for sufficiently long polyelectrolytes; further, differences in
the mobility of large ionic polymers due to different MW values should be
obtained only upon addition of a sieving medium to the BGE [Cottet, 2005].
As shown below in section VI.2.1.8, the peak areas of the complexes
increased linearly with the monomer concentration, but were essentially
independent of MW. However, as observed in Figs. VI.11, the shape of the peak
of the PVPdye complexes varied with MW. In fact, the height/width ratio of the
peaks increased with both MW and the PVP concentration. Thus, a method to
226
w1/2
2
-0.5
Time (min)
-1.5
1.2
2.2
3.1
3.6
4
-2.5
4
4.5
log MW
5.5
Fig. VI.12. Logarithmic plot of (h/w)/CPVP against MW; h and w are the
height and base width of the peak of the PVPCR complexes, which were
measured as indicated in part A (w =2w1/2). The units used were mAU, min
and mg mL1 for h, w and CPVP, respectively. The dashed lines indicate
confidence limits for a significance of 0.05. The legend in part B indicates
the q values of the series. Other details as in Fig. VI.11.
227
a reduced dispersion of the points are observed. This relationship between peak
shape and MW could be due to the increase in the stability of the complexes as
MW increased. However, another possible reason could be an increase in the
polydispersity of the PVPdye complexes as MW decreased.
228
A
200
500
100
60 kDa PVP
0
3
Relative Ae + AD values
300
B
2
27.9 kDa PVP
60 kDa PVP
400 kDa PVP
0
0
0.2
0.4
0.6
1/q (reverse of the monomer/ dye ratio)
Fig. VI.13. Area (APVPD, rhombus and continuous line) (A) and
electrophoretic mobility (B) of the peak of the PVPCR complexes at
increasing CR concentrations, keeping a constant monomer concentration
of 4.5 mM. In A, the squares and dashed lines correspond to Ae + AD (right
scale). The MW values are indicated on the plots. The electropherograms
were recorded at 500 nm.
Next, the PVP concentration was increased, while the dye concentration
was maintained constant. As shown in Fig. VI.14, part A for the CR complexes
with 60 kDa PVP, the peak area of the complexes increased at increasing q
values. This plot, and similar plots obtained with PVP having other MW values, as
well as by using AB, also indicated the formation of a complex with a maximal
monomer: dye molar ratio of q 4. A difference with respect to Fig. VI.13, part
A, was the linearity of the relationship between APVPD and the PVP
concentration, at least up to q = 3.5. Linearity was attributed to saturation of the
229
polymer in the presence of an excess dye in this region of the curves. As shown
later in this work, this was of interest for the CE determination of the polymer.
Another feature of Fig. VI.14, part A, which was not observed in Fig. VI.13,
part A, was an abrupt increase of the peak area of the PVPCR complexes
immediately before reaching the saturation point.
800
1000
A
400
60 kDa PVP
0
3.0
Relative Ae + AD values
1200
2.5
2.0
0
This could be due to a higher stability of the complexes in the vicinity of the
saturation point, where a more favourable conformation could exist; however, a
slower dissociation rate of the complexes could also contribute. As also observed
in Fig. VI.14, part A, the remaining area, (Ae + AD), decreased at increasing PVP
230
231
5002000 g mL1 PVP, and at 1/q values equal to zero, 0.05 and 0.1 were
performed. These series were monitored at 215 nm. The curves were fitted to the
cubic equation, and for each curve, the initial slope was obtained as the
regression coefficient of the first grade term. A plot of the initial slopes against
log MW is shown in Fig. VI.15. Although with some dispersion, this plot showed
a decrease in the initial slopes when log MW increased. The possible reasons of
/ (1/q) at (1/q) 0
200
100
0
4
log MW
Fig. VI.15. Initial slopes of the curves in Fig. VI.13, part B (variation in
the mobility of the complexes vs. 1/q) plotted against log MW. Data
obtained as indicated in the text.
the
opposite
behaviour
was
actually
observed
when
232
[ D ] eq =
AD
b D
(E.VI.1)
where bD is the optical path multiplied by the molar absorptivity of the dye. The
equilibrium molar concentration of the complexes is dependent on APVPD and Ae
as follows:
[ PVP D ] eq =
APVP D
A
+ e
b PVP D b D
(E.VI.2)
where PVPD is the average molar absorptivity of the complexes, which should be
calculated with reference to the molar concentration of the complexed dye. This
is equivalent to considering that a dye ion is complexed on a 1:1 basis by a group
formed by a fixed number of monomers, complexing polymer units, or binding
sites. In this way, the possible influence of a stoichiometry different from 1:1 is
ruled out. The ratio of the two equilibrium molar concentrations is:
233
R=
[ PVP D ] eq
[ D ] eq
APVP D ( D / PVP D ) + Ae
AD
(E.VI.3)
1+ R
[C ] 0 (1 + 1 R) [ D ] 0
(E.VI.4)
where [C]0 and [D]0 are the analytical molar concentrations of the complexing
polymer units and the dye, respectively. Since the maximal stoichiometry of the
PVP complexes with CR and AB is 4:1, we have:
N
4
[C ] 0 = [ PVP ] 0
(E.VI.5)
234
10
27.9
40
60
360
3.1
4.350.05
4.290.01
4.330.01
4.300.01
4.300.01
4.450.02
3.6
5.010.05
5.040.05
5.220.03
4.170.71
5.460.07
4.0
5.040.03
4.940.12
5.23*
5.430.12
4.730.14
5.660.06
4.5
4.670.08
4.350.01
5.09*
4.990.11
4.820.15
5.230.11
400
In addition, the log Ka values given in Table VI.2 also showed that the stability
of the complexes when q = 4 increased with MW. Finally, it should be noted that
owing to the Joule heating, the values in Table VI.2 were obtained at a capillary
temperature which was actually higher than 25C [Evenhuis, 2009].
235
a)
MW (kDa)
CR
AB
10
0.94
0.87
27.9
0.95
0.96
40
1.07
1.01
60a
1.00
1.00
160
1.54
1.57
360
0.92
0.93
400
1.28
2.00
Taken as reference.
236
Absorbance (mAU)
150
100
20
10
0
3.0
Time (min)
4.0
A
50
215 nm
B
500 nm
0
0
Time (min)
237
[Prudhomme de Lodder,
The concentrations found for the cough syrup and the antibacterial tablet
were well within the usual ranges, namely 0.55% as binders in tablets and <5%
for dispersing agents in syrups [Rowe, 2005]. Finally, the declared amount of PVP
in the topical antiseptic was 10%. Then, the plot of Fig. VI.12, part B was used
to predict the MW values of Table VI.4. Values below 35 kDa were predicted for
the laundry cleaners. Owing to the interference of the surfactants, these values
were probably lower than the actual values. A large value (MW = 400 kDa) was
obtained for the cough syrup, which was attributed to the presence of the anionic
azo-dye amaranth (E123). This dye could form also complexes with PVP with a
mobility similar to that of the PVPCR complexes, thus contributing to the peak
area. Finally, MW = 22 and 16 kDa were predicted for the antibacterial tablet and
topical antiseptic, respectively. The actual MW values in these products are to be
expected along wide ranges [Rowe, 2005].
Table VI.4. PVP concentrations predicted using external calibration with 60 kDa
PVP and MW values estimated by applying the plot of Fig. VI.12, part B.
MW (kDa)
Concentration (%wt)
MW (kDa)
Laundry cleaner I
0.89
35
Laundry cleaner II
0.31
10
0.45
<10
Cough syrup
0.95
400
Antibacterial tablet
2.42
22
Topical antiseptic
12.4
16
238
VI.3. PVA
[Clos, 1998; Grosche, 2000; Borisch, 2000; Engelhardt, 2004; Cottet, 2005].
Using CE, information about size, shape, surface charge and formation of
intramolecular
associates
can
be
gained
[Engelhardt,
2004].
Further,
[Long,
and CZE has been also used to study both the polymerization degree and
the sulfonation rate of polyestyrenesulfonates [Cottet, 2000]. MECK has been used
to characterize highly charged polysaccharides (heparins)
polyacrylic acids
[Collet, 1996],
[Stefansson, 1994],
and
[Beneito-Cambra, 2009-A],
In a
characterize and evaluate PVP; for this purpose, we used the azo-dye CR which
forms a charged and colored PVPCR complex. The electropherograms of PVP
CR mixtures were interpreted at the light of the NECEEM theory, which was
developed by Krylov et al.
239
mixtures and the BGE, the expected NECEEM pattern for a mixture of a noncharged macromolecule and a charged marker was obtained. Commercial PVA
samples with different molecular masses, also differing in tacticity, were studied.
The electropherograms of PVACR mixtures provided information about the
electrophoretic mobility, maximal stoichiometry, thermodynamic stability
constant and pseudo first-order dissociation rate constant of the PVACR
240
complex. These parameters were observed to depend on both molecular mass and
tacticity of PVA.
[Fraunenfelder,
As indicated in section
III.2.3.3, the formation of the complexes was first studied by filling the capillary
with PVACR mixtures containing 20 mM borax. As illustrated in Fig. VI.17 for
the 49 kDa PVA sample, the UVVis spectrum of a 4 mM CR solution was
largely
modified
when
the
mixture
also
contained
increasing
PVA
concentrations.
q=6
q=9
Absorbance (mAU)
600
400
q = 12
q=3
200
q=0
0
300
400
500
600
Wavelength (nm)
Fig. VI.17. UVVis absorption spectra obtained by filling the capillary
with solutions containing 20 mM borax, 4 mM CR and the following PVA
concentrations (49 kDa): 0, 12, 24, 36 and 48 mM (the resulting q values
are indicated on the traces).
shift of about 40 nm was also observed (from 479 to 519 nm). Similarly, an
intensity increase of ca. 55% and a bathochromic shift of ca. 50 nm (from 489 to
539 nm) were observed with a conventional spectrophotometer when 0.4 mM
PVA was added to a 0.1 mM CR solution (spectra not shown). This implies a
major change in the physico-chemical environment of CR [Olsen, 1975]. The large
modification of the CR spectrum could be partially due to replacing of water
molecules attached to the polar locations of CR by the polar groups of the
polymer; however, as discussed below in section VI.3.1.4, another possible
reason is the stacking of dye ions in proximity to each other within the structure
of the PVACR complex. An absorptivity decrease at all wavelengths was also
observed when q > 6. This could be due to an increase of the refraction index of
the solution or to the optical screening of the dye in the presence of a large
polymer excess.
242
[Berezovski, 2002; Krylov, 2003; Drabovich, 2006; Lin, 2008; Krylov, 2006;
Krylov, 2007].
the PVACR complex, and the peak that followed was assumed to be contributed
by both the initial CR excess in free form and the dye released by the complex
during migration (contributing mainly to the left half of the peak). As q increased
(from traces A to D), the area of the band due to the PVACR complex
increased, and the area of the peak due to the free dye decreased. Above q 4 the
electropherograms showed the single band of the complex, which progressively
widened when q further increased (trace D). In addition, a BGE containing 20
mM Na2HPO4 (pH 10) instead of borax was tried. Mixtures containing 4 mM CR
and 16 mM PVA (q = 4) were injected; however, a low intensity wide band due
to the PVACR complex, and a large peak and exponential decay due to the free
dye, were observed (electropherograms not shown). Thus, owing to the better
shape and higher intensity of the complex band, the BGE containing a borax
excess was preferred.
The time required to equilibrate the mixtures before injection was studied.
For this purpose, aliquots of a solution containing 20 mM borax, 4 mM CR and
16 mM PVA monomers (49 kDa, q = 4) were injected at regular time intervals
after mixing the reagents. The area due to the remaining complex, APVA-D, was
estimated as indicated in Fig. VI.18, traces A to D, by doubling the area of the
left half of the complex band. The rest of the area, due to both the initial free dye
and the dye released by the complex during migration, was measured as
ATotalAPVA-D. A slow increase of APVA-D (ca. 8%), a decrease of the rest of the
area (ca. 20%), and a small increase of the electrophoretic mobility of the
complex (ca. 4%), were observed during the first 2 h after mixing the reagents
(sequence of electropherograms with time, not shown). This suggested a slow
243
300
APVA-D / 2
EOF
q=1
200
q=2
q=3
D
E
F
q=9
100
0
0
4
Time (min)
samples. As shown in Fig. VI.19 for the 15 kDa PVA sample, when q was
increased the area of the PVACR complex, APVA-D, increased linearly up to q
4, and the rest of the total area decreased proportionally. When q 5, that is,
above the saturation point, the peak due to the excess dye was not observed any
longer, and APVA-D decreased steadily. This agreed with the decrease in the
absorptivity of the complex which was observed by recording spectra at large q
values (Fig. VI.17).
100
Atotal APVA-D
APVA-D
0
0
4
6
q = [monomer] / [CR]
10
The other PVA samples behaved similarly, although with differences in the
location of the saturation point indicating the maximal stoichiometry of the
complex. In order to establish the saturation point of PVA complexes, qsat, as
accurately as possible, mixtures with q values close to the expected qsat values
245
4.5
4.0
3.5
1.5
2.0
Log MW
Fig. VI.20. Plots of the maximal stoichiometry of the complex against log
MW without (qsat, full symbols) and with correction for the acetyl
percentage (qsat,c, empty symbols). Symbols indicating tacticity groups: H
(diamonds), M (circles) and L (triangles).
246
Next, a correction of the qsat values, thus to take into account the different
proportions of residual acetyl groups in the PVA samples was tried. The molar
proportions of residual acetyl groups per mol of monomers, nac, for the PVA
samples used in this work, were given in section III.2.3.1. In a PVA molecule,
acetyl groups could reduce the number of OH groups which are actually
available to link the dye ions. Thus, in nac 100% acetylated PVA, only
(1nac)100% of the monomers would be available for bonding; however, this
should not reduce the complexing capacity of PVA in an equivalent
(1nac)100% factor, since acetylated monomers can also perform as
nonbonding bridges between bonded monomers. Nevertheless, we studied next
the effect which would have on qsat the extreme situation in which the
complexing capacity of PVA would be reduced in a full (1nac)100% factor.
Thus, the correction applied was: qsat,c = qsat/(1nac). As shown in Fig. VI.20
(empty symbols), full correction for the acetyl percentage essentially confirmed
the conclusions obtained above using uncorrected qsat values concerning both the
decrease of qsat at increasing molecular masses and the noticeable difference
between the H and M+L tacticity groups.
247
ion is very low, which suggests a proximity between the dye ions. As depicted in
the temptative structure of Fig. VI.21, stacks of dye ions by pairs, or by groups
constituted by a higher number of dye ions, could be formed. As proposed in this
figure, it seems reasonably to stack the dye ions by alternating the sulfonate and
amino groups, rather than putting together all the sulfonate groups at one side
and all the amino groups at the other side of the stack. Also, from a geometrical
point of view, it seems reasonably to link the PVA monomers with the alternated
sulfonate and amino groups though hydrogen bonds. As indicated in the
literature, the azo groups are also possible sites for hydrogen bonding
2001; Olsen, 1975.];
[Atkin,
stoichiometry, the number of available monomers per dye ion is rather short.
Then, hydrogen bonding of the PVA monomers with both amino and azo groups,
which are located in outer and inner sites of the dye ion, respectively, seems to be
less likely than the structure proposed in Fig. VI.21 However, the probability of
bonding the OH groups with azo groups should increase after the saturation
point, when an excess of monomers are available.
Dye stacking, resulting in a large absorptivity increase and blue shifting of
the absorption maximum, has been described in solutions of plant pigments when
certain ligands and Mg2+ are present [Ellestad, 2006]. Thus, dye stacking could
explain both the low qsat values of Fig. VI.20 and the very large modification of
the absorption spectrum when PVA is added to CR solutions (Fig. VI.17).
Further, the distance between donoracceptor atoms in adjacent stacked dye ions
should be constricted by the distances between pairs of adjacent OH groups
along the PVA chain, and these later are longer for r than for m diads. Thus, in
PVA samples having a larger proportion of r diads, adjacent dye ions could be
stacked at longer distances from each other than in samples with larger
proportions of m diads. As discussed below, this could also explain the
248
O
H
O
H
H
N
H O
N
H
N
N
O
N
H
H
H
OH
O-
O H
O-
O
S N
O
N
N
O-
H
H
O-
O
H
H
O
O-
H
H
H
O
Fig. VI.21. Temptative structure for two adjacent PVACR complex units
(with two stacked dye ions).
249
250
volume ratio which is achieved when units with a given charge-to-volume ratio
are increasingly added to the polyelectrolyte. This electrophoretic behavior of
polyelectrolytes has been called the free draining regime [Cottet, 2000].
APVA-D / 2
200
AD
EOF
4; 4.5
AD
150
3; 3.5
A
D
4; 4.5
100
A
D
3; 3.5
50
AD
3; 3.5
0
0
4
6
Time (min)
10
251
60
30
50
40
25
1.0
1.5
Log MW
2.0
252
attached to that ion, plus the average share of monomers which are necessary to
link the complexed dye ion to the neighboring complex units. The mobility per
complex unit, uni, should be directly proportional to the charge of a dye ion, 2,
and inversely proportional to the volume of the complex unit:
unit =
2f
VD + qsatVm
(E.VI.6)
(E.VI.7)
253
corrected mobilities of Fig. VI.23 also indicated that the complex did not reach a
free draining regime at increasing molecular masses. This meant that coefficient f
in equation E.VI.7 was not constant within any molecular mass range, that is,
packing density of the complex decreased at increasing molecular masses for all
the PVA samples used in this work.
254
dye migrates towards increasing migration times). Then, the area at the right of
the first depression of the free dye peak (see the expanded parts in Fig. VI.22)
was assigned to AD, and Ae was calculated as the total area minus the
contributions of the complex and initially free dye: Ae = ATotal (APVA-D + AD).
These assignments probably implied a systematic error in the estimation of AD,
and therefore also in the calculation of the stability constants; however, this made
possible to compare the constants obtained with the different PVA samples. To
estimate stability constants, the equilibrium molar concentration of free dye,
[D]eq, was obtained as:
[ D ] eq =
AD
b D
(E.VI.8)
where bD is the optical path multiplied by the molar absorptivity of the dye. The
equilibrium molar concentration of the complex is given by:
[ PVA D ] eq =
APVA D
A
+ e
b PVA D b D
(E.VI.9)
where PVA-D is the molar absorptivity of the complex. Instead of reasoning about
complex formation in terms of qsat monomers per dye ion, calculations are much
simpler if the formation of a 1:1 complex is assumed, being the ligand the
complexing units formed by groups of qsat monomers. The stability constant, Ks,
is then given by:
Ks =
1+ R
[C ] 0 (1 + (1/ R)) [ D ] 0
(E.VI.10)
where [C]0 and [D]0 are the total analytical molar concentrations of the
complexing polymer units and the dye, respectively, and where R is given by:
R=
[ PVA D ] eq
[ D ] eq
255
APVA D ( D / PVA D ) + A e
AD
(E.VI.11)
[C ] 0 =
n
[ PVA] 0
q sat
(E.VI.12)
where n is the average number of monomers along the polymer chains, and
where [PVA]0 is the total analytical molar concentration of the polymer in mol
L1. The molar absorptivity ratio D/PVA-D = 0.77 was obtained with a
spectrophotometer at 500 nm by measuring several PVACR mixtures with q
values sligthly lower than the respective qsat values; in these conditions, the
formation of a predominant complex with a 1:1 stoichiometry, in which the
ligand was constituted by qsat monomers, was assumed. In Fig. VI.24, part A,
the resulting values of log Ks for q = 2 and 3 (full and empty symbols,
respectively) were plotted against log MW. Contrary to that observed for other
CZE parameters, log Ks values did not show any significant trend concerning to
either molecular mass or tacticity.
The electropherograms with q = 2 used to obtain log Ks were further
processed to estimate pseudo-first-order rate constants for the dissociation of the
PVACR complexes. For this purpose, half-life measurements were made, and
rate constants were estimated as k = ln2/t1/2, where t1/2 is the half-life. The
exponential decay curve within the region close to the peak of the excess dye,
where the contribution of the remaining PVACR complex was small, was used.
Five measurements of the half-life were made by selecting 5 different starting
points on the decay curve of each electropherogram of series of triplicated
injections. The starting points were evenly spaced from each other in about 5 s.
Estimations of k were obtained with low uncertainties (ca. 4.3%). In Fig. VI.24,
part B, the average values of k were plotted against log MW. Within each tacticity
group, the dissociation rate constant of the complex was reduced at increasing
molecular masses; in addition, H samples showed differences with respect to the
M+L samples.
256
5.0
4.0
3.0
2.0
1.0
5.0
4.0
3.0
2.0
1.0
1.0
1.5
2.0
Log MW
Fig. VI.24. Plots of (A) log Ks and (B) k vs. log MW. Data were calculated
from electropherograms obtained with (A) q = 3 and 2 (full and empty
symbols, respectively) and (B) q = 2. Symbols indicating tacticity groups:
H (diamonds), M (circles) and L (triangles). Other conditions as in Fig.
VI.22.
257
CHAPTER VII
ENZYMES
[Novozymes,
2002].
Since the
the role of enzymes in cleaning products has changed from that of a minor
[Olsen, 1998].
products constitute the largest segment of the world market for industrial
enzymes
[Novozymes, 2002].
[www.heraproject.com.],
[Deschreider,
1972].
Protein
261
or with a
silylating reagent [Rampazzi, 2002], and a wide variety of HPLC methods [MolnrPerl, 2000; Molnr-Perl, 2001; Tcherkas, 2001-A; Tcherkas, 2001-B; Concha-Herrera,
2007; Hanczk, 2007; Chernobrovkin, 2007; Pereira, 2008].
[Beneito-Cambra, 2008;
[Jmeian, 2009;
[Ruiz-ngel, 2002],
[Righetti, 1997; Bossi, 1997; Righetti, 1998; Capelli, 1998; Stoyanov, 1997]
and
HEC to inhibit adsorption, was also used. Due to their much reduced
conductivities, large concentrations of isoelectric buffers are compatible with
high voltage gradients, thus further reducing adsorption and favouring high
resolution within short migration times
Capelli, 1998].
by the Bradfords method. This made possible to use total peak areas of the
electropherograms obtained with both BGEs to establish relative sensitivities,
which were also useful for enzyme identification. The enzymes used in this work
are indicated in Table III.2.
A
Absorbance at 214 nm (mAU)
Esp
Sta
EOF
800
EOF
400
120
EOF
80
Cel
40
Car
Lx
Eve
80
200
400
Dur
40
Sav
EOF
Ls
End
Ter
Alc
0
2
0
2
Time (min)
For all proteases (Fig.VII.1, part A), the main peak was located within the
cationic migration region in the vicinity of the electroosmotic flow (EOF) time
(ca. 2.14 min). At pH = 9.0 charge density should be low for proteases (8.4 < pI
< 11), which agrees with the cationic behaviour of the main peak. The other
263
enzymes (Fig.VII.1, parts B to D), with the exception of Sta, gave also an
intense peak or a group of peaks within the anionic migration region. Sta gave
two partially resolved peaks close to the EOF time, but within the cationic
region. An amylase, Ter, and a lipase, Lx, showed two peaks of similar intensity,
with baseline resolution, instead of a single predominant peak (Fig.VII.1, parts
B and C). The presence of a second intense peak close to the EOF time made Ter
to be easily distinguishable from its engineered variant Dur (Fig.VII.1, part B).
Similarly, the sharp intense peak within the cationic region made Lx to be easily
distinguishable from the other lipase, Ls (Fig.VII.1, part C). Also, some
cellulases as Cel and End (Fig.VII.1, part D) showed a division of the main
band into several partially resolved intense peaks. In addition to the main peaks,
the electrophoretic profiles of most enzymes also provided several small peaks
which can be useful as fingerprints for identification.
Intra- and inter-day repeatabilities in this BGE were obtained by injecting
a 0.01% Alc aqueous solution. Five injections per day during three consecutive
days were performed. Intra- and inter-day migration times showed RSDs lower
than 3.5 and 7.5%, respectively. More than 100 injections were performed
without the need of replacing the capillary.
264
sharp peak at ca. 3.5 min. The presence of two large peaks in these two enzymes
could be due to the occurrence of two predominant protein fractions in the raw
material. It should be noted that Esp and Sav are two closely related proteases
with well known three-dimensional structures, and a high degree of structural
similarity
[Georgieva, 2001].
Sta
60
120
200
Cel
Esp
40
Dur
40
80
Eve
100
Lx
Car
20
Sav
20
40
Ls
End
Ter
Alc
0
0
4
0
4
Time (min)
265
gave a single asymmetric peak. Using Alc, migration time intra- and inter-day
repeatabilities were below 2.7 and 5.3%, respectively. At least 120 injections
were performed without the need of replacing the capillary. Therefore, both in
the basic and acid BGEs, most enzymes gave characteristic patterns which
allowed class identification, and in many cases the electropherograms also
showed enough distinctive details to allow the safe identification of the
individual enzyme within its class.
Class
Protease
Amylase
Lipase
Cellulase
Relative
sensitivityc)
Basic
Acid
BGE
BGE
1.0
1.0
Alcalase 2.5 L
Alc
Protein content
(% BSA)b)
4.03 0.03
Sav
1.81 0.07
1.8
1.7
Eve
1.24 0.03
2.8
1.0
Esperase 8.0L
Esp
2.45 0.05
1.5
1.4
Ter
2.95 0.02
1.9
1.1
Dur
4.87 0.03
0.8
0.3
Stainzyme 12L
Sta
2.69 0.01
1.0
0.9
Ls
1.79 0.01
1.2
1.2
Lipex 100L
Lx
2.90 0.01
0.8
0.7
Endolase 5000L
End
2.56 0.01
1.1
1.0
Carezyme 4500L
Car
0.74 0.08
1.5
1.4
Celluzyme 0,7Ta)
Cel
1.31 0.06
4.9
0.4
Commercial name
Abbreviation
a)
Sample supplied as granular solid (the other samples were liquid concentrates)
Estimated using the Bradfords assay (calibration curve with 9 points)
c)
Relative sensitivities for the total area of the electrophoretic peaks (respect to Alcalase 2.5L)
b)
266
Then, for each industrial enzyme a calibration curve was constructed. For this
purpose, industrial enzymes were diluted with water at several concentration
levels within the 0.2-20% v/v range, and aliquots were taken. Precipitation with
acetone, re-dissolution and injection in the capillary using both the basic and acid
BGEs were then performed. The plots of total peak area versus protein
concentration were linear (r > 0.992). From these plots, relative sensitivities were
established as follows:
S r = S x S Alc
(E.VII.1)
where Sx and SAlc are the slopes for the assayed enzyme and Alc which was used
as reference, respectively. As shown in Table VII.1, the ranges were 0.8-4.9 and
0.3-1.7 for the basic and acid BGEs, respectively. The differences between
relative sensitivities were small in a number of cases, but several enzymes gave
rather characteristic values. Thus, Eve and Sav gave high sensitivities in the basic
and acid media, respectively, Dur gave low sensitivities in both media, and Cel
exhibited a very high sensitivity in the basic BGE but a very low one in the acid
BGE. As also deduced from Table VII.1, the differences among relative
sensitivities were predominantly due to the nature of the individual enzymes
rather than to their respective classes.
267
part B); however, rather different profiles were obtained when the sample
solution contained anionic surfactants (Fig. VII.3, parts C and D). Similar
experiments were performed in the acid BGE. The influence of Dehydol LT-7
gave electropherograms similar to that of Fig. VII.2, part A for Alc; however, in
the presence of anionic surfactants, a significant increase in the retention time of
the enzyme peaks was observed (data not shown). Similar results were obtained
for selected enzymes of the other three classes. Consequently, the proposed
method for enzyme identification can be safely applied in the presence of nonionic surfactants, but will fail probably when anionic surfactants would be
present at large concentrations. Work addressed to reduce the interference of
anionic surfactants is in progress.
80
80
40
40
0
20
0
20
C
10
10
0
0
Time (min)
Fig. VII.3. Influence of surfactants on the electrophoretic profile of Alc
(0.01%) in the basic BGE: (A) absence of surfactant, (B) 5% Dehydol LT7, (C) 5% LAS and (D) 5% LES. Other conditions as in Fig. VII.1.
268
[Novozymes, 2002].
Since
then, the role of enzymes in cleaning products has changed from one of a minor
additive to becoming a key ingredient [Olsen, 1998]. Cleaning power enhancement
by enzymes leads to a reduction of washing times and temperatures, with the
subsequent savings of water and energy. Further environmental advantages arise
from the lower consumption of surfactants, hypochlorite and alkalis, with the
additional benefit of a better care of fabrics during washing [Olsen, 1998].
Mainly proteases, amylases, lypases and cellulases are used in the
formulation of modern cleaning products. Proteases are used to remove proteinrich soil stains, including blood and grass. Amylases remove starch-containing
stains, and prevent starch from adhering to fabrics and dishes. Lypases help in
removing fat and edible oil stains. Finally, cellulases are used to remove fuzz and
pills (small balls of fabric) from cotton. In spite of the importance of these
enzymes in the detergent industry, and in environmental and toxicological studies
[www.heraproject. com],
269
classification. After hydrolysis, the amino acid profile of the enzyme can be
established by a variety of separation and detection techniques, including GC
following previous derivatization with ethyl chloroformate
2002; De la Cruz-Caizares, 2004],
[Gimeno-Adelantado,
[Rampazzi, 2002],
[Goodacre, 2002;
or an APPI
rice cultivars
[Wang, 1998],
[Poulli, 2005],
LDA
partial least-squares
[Erbe, 2000].
[Lerma-Garca, 2007],
[Alczar, 2007],
as well as to
hierarchical cluster
and ANN
[Garca-Gonzlez, 2004],
have
270
1.2
Trp
Tyr
Arg
Phe
Asn
Asp
Lys
Glu
Met
His
Thr
Cys
Ala
Ala
Ser
Gly
0.4
Gly
Cys
Met
Ser
0.8
Val
Pro
Leu+Ile
0.05
0
80
160
120
m/z
271
200
272
273
0.022), and the number of predictors selected from the 153 variables initially
established decreased from 18 to 13. The predictors and their standardized
coefficients for the three discriminant functions obtained by using Fin=0.05 and
0.01 are shown in Table VII.2. Using normalization procedure B, the ion
abundance ratios of pairs of amino acids rather than the ion abundances of
individual amino acids are used as predictors.
Table VII.2. Predictors selected and standardized coefficients of the LDA models
constructed by jointly using training matrices M1B (enzyme industrial concentrates)
and M2B (detergent bases spiked with enzyme industrial concentrates) with two
different entrance thresholds.
Fin=0.05
f2
f3
6.34 -0.800
f1
Fin=0.01
f2
Selected variable
Val/Met
f1
3.44
f3
Val/Thr
-2.78
0.715
5.71
1.92
0.51
5.82
Leu+Ile/Met
-2.12
-0.744
-0.22
0.295
1.70
-0.326
Phe/His
3.24
0.805
0.92
-4.10
0.839
1.04
Phe/Lys
-6.78
12.1
3.74
3.02
2.57
3.75
Phe/Arg
-6.43
4.47
-2.02
6.41
0.172
-2.72
Phe /Ser
2.06
0.849
0.070
-1.67
-0.247
-0.237
Phe/Trp
3.08
-3.20
-0.261
-3.43
-2.16
-0.572
Phe/Cys
-1.63
-4.76
-0.424
Phe/Pro
1.78
-1.34
-2.07
-0.927
0.698
-1.22
Glu/His
-1.07
-1.70
3.69
1.85
0.275
4.21
His/Arg
0.95
-2.95
-0.831
-2.47
-0.545
-0.254
Lys/Arg
10.6
-17.0
-5.19
-5.17
-2.41
-3.99
Lys/Trp
-8.26
15.5
2.02
2.42
2.11
0.646
Gly/Trp
-1.21
0.846
0.426
Ser/Thr
0.513
-1.19
0.088
Thr/Trp
5.75
-4.06
-0.255
Tyr/Pro
-0.158
-3.63
0.022
Phe/Thr
2.14
0.290
-0.002
274
suggested that Phe was important in distinguishing enzyme classes, and that the
pairs Lys/Arg, Lys/Trp, Phe/Lys and Phe/Arg, among others, were also
particularly important.
10
20
A
lipases
10
proteases
cellulases
amylases
-10
-60
proteases
lipases
0
cellulases
-10
amylases
-20
-10
-40
-20
0
20
First discriminant function
0
10
20
30
Second discriminant function
C
First discriminant function
30
proteases
lipases
20
0
-20
amylases
cellulases
-40
30
10
20
10
0
-10
Fig. VII.5. Score plots on the planes of the first and second (A), and
second and third discriminant functions (B), and on an oblique plane of the
3-D space defined by the three discriminant functions (C). Matrices M1B
(industrial concentrates of enzymes selected for training) and M2B (two
detergent bases spiked with the same industrial concentrates of enzymes)
were jointly used to construct the LDA model. The predictors were
selected using Fin=0.01.
275
[Novozymes, 2002].
276
[Olsen, 1998],
with the
[www.heraproject.com],
identification and
[Olsen, 1998;
[Gimeno-
2001; Tcherkas, 2001-A; Tcherkas, 2001-B; Garca-lvarez-Coque, 1989; ConchaHerrera, 2007; Hanczk, 2007; Chernobrovkin, 2007; Pereira, 2008].
Among these,
[Concha-Herrera, 2007],
fluorimetry
[Molnr-Perl,
or electrochemical techniques
[Tcherkas,
and ethanethiol
[Hanczk, 2007],
[Wang, 1998].
and LDA
vegetable oils
[Lerma-Garca, 2007],
[Alczar, 2007]
[Llet, 2003]
[Concha-Herrera, 2006],
Alc. First, the OPA concentration was increased, while both an OPA/NAC molar
ratio of 1:2 and an OPANAC/hydrolyzate ratio (v/v) of 10:1 were maintained at
fixed values. The peak areas increased when the OPANAC concentration
278
[Concha-Herrera, 2006]
to 1.25102 M. Then,
[www.cas.org/SCIFINDER],
was
selected. Owing to the increase of the dilution factor, the peak areas were
reduced to ca. 50% when the OPANAC volume was increased from 1 to 2mL,
while maintaining an hydrolyzate volume of 100 L. The amino acid peak areas
also decreased when the hydrolyzate volume was increased to 0.5 mL, which
could be due to a reduction of the reaction yield at the decreasing pH of the
mixture. Thus, further studies were performed with an OPANAC/hydrolyzate
ratio of 10:1 (v/v). Under these conditions, the other enzymes of Table III.2,
section III.3.1.1 also provided satisfactory peak intensities.
To optimize the chromatographic separation of the amino acids, the multisegmented gradient of Concha-Herrera et al.
[Concha-Herrera, 2006]
was initially
used. This consisted of three linear steps where the ACN concentration was
increased as follows: 518.5% (30 min), 18.522% (40 min) and 2227.5% (10
min). However, with this gradient a few peaks overlapped at long retention times.
Then, careful trial-and-error optimization of the multi-segmented gradient was
carried out in order to improve resolution between the critical peak pairs. With
the gradient described in Table VII.3, and as illustrated in Fig. VII.6, all the
amino acid peak pairs, except the Phe/Leu pair, were baseline resolved.
Table VII.3. Optimal multi-segmented gradient accomplished by mixing 5% (A)
and 50% (B) ACN/water solutions, both buffered at pH 6.5 with 5mM sodium
citrate/citric acid.
Time (min)
A(%)
B(%)
100
28
72.5
27.5
36
70
30
42
44.4
55.6
50
42.4
57.6
55
100
279
Phe + Leu
Thr
30
Ala
Asp
80
20
Val
ACN %
120
Glu
40
His
10
Tyr
Ser
Gly
Arg
Ile
Lys
Met
0
0
10
20
30
40 Time (min)
50
Also, the Ser peak, which was well resolved in the chromatogram of Fig.
VII.6,was only partially resolved from an unidentified peak (see Fig. VII.6) in
the chromatograms of a few other enzymes. Thus, to construct LDA models, the
Ser peak was not used, and the Phe/Leu peak pair was jointly measured, 13 peak
areas corresponding to 14 amino acids being then selected.
VII.3.1.2. Data matrices and construction and evaluation of the LDA models
In order to reduce the variability associated to the total amount of protein
recovered from the samples and to their hydrolysis, normalized rather than
absolute values of the peak areas were used. For this purpose, two normalization
procedures were tried. In procedure A, the area of each amino acid peak was
divided by the sum of the areas of all the amino acid peaks of the chromatogram.
In procedure B, the area of each amino acid peak was divided by each one of the
280
areas of the other 12 amino acid peaks; in this way, and taking into account that a
pair of peaks should be considered only once, (1312)/2=78 non-redundant ratios
of peak areas were obtained.
To construct LDA models for enzyme class prediction, the training set
was first constituted by the enzyme industrial concentrates indicated in Table
III.2, section III.3.1.1. Three enzymes from each one of the four enzyme classes
were selected. As indicated in section III.3.3.4, two aliquots of each enzyme were
hydrolyzed and injected; however, only the average of the peak areas of the two
injections was included in the training matrix. In this way, the internal variance
of the categories was reduced, which was important to also reduce the number of
variables selected by the stepwise algorithm during model construction.
Therefore, the training matrices were initially constituted by 12 objects each (3
enzymes 4 enzyme classes 1 average of two hydrolyzates) and either 13 or 78
variables obtained according to normalization procedures A and B, respectively.
The resulting LDA models were used to predict the enzyme class in the
two detergent bases spiked with the four enzymes indicated in Table III.2. Data
obtained from a total of 16 chromatograms were evaluated (4 enzyme industrial
concentrates 2 detergent bases 2 hydrolyzed aliquots of each mixture).
However, the two models showed a poor prediction capability (2530% of
correct assignments for a 95% probability level) which was attributed to the
matrix effect produced by the anionic surfactants and other components present
in large concentrations in the detergent bases. Thus, in order to increase the
prediction capability of the models, data obtained with the spiked detergent
baseswere also included in the training set. Thus, the expanded training matrices
had 20 objects (3 enzyme industrial concentrates plus 2 spiked detergent bases
4 enzyme classes 1 average of two hydrolyzates), and either 13 or 78 predictors
as indicated. Then, the duplicate hydrolyzates of the enzyme industrial
281
concentrates of Table III.2 which were not included in the training set, plus the
duplicated hydrolyzates of the spiked detergent bases and the two commercial
cleaners, were used to construct the evaluation matrices. Thus, the two evaluation
matrices had 58 objects each (19 industrial concentrates of enzymes plus 8
spiked detergent bases and 2 cleaning products 2 hydrolyzates each).
Using normalization procedure A, an LDA model with 9 variables,
showing an excellent resolution between all the possible pairs of categories (W =
0.032), was obtained. However, only a 30% of the samples of the evaluation
setwere correctly classified by this model. On the other hand, using
normalization procedure B, an LDA model with 8 variables, showing a slightly
better value of W (0.019) than the previous model (0.032) was obtained. Further,
all the 58 samples of the evaluation set were correctly classified, with assignment
probabilities higher than 99%. The predictors and respective standardized
coefficients of the three discriminant functions of this model are shown in Table
VII.4. The use of ratios of areas of peak pairs as predictors, rather than individual
peak areas, prevented from reliably assigning any particular relevance to the
discriminant capability of individual amino acids.
Table VII.4. Predictors and their corresponding standardized coefficients of the
optimal LDA model.
f1
f2
Asp/Val
0.92
0.56
-0.55
Glu/His
0.63
-0.76
1.76
Glu/Ala
0.07
1.04
0.82
His/Thr
0.20
-3.77
2.29
Thr/(Leu+Phe)
1.09
0.25
1.46
Ala/Tyr
-0.45
2.35
-1.70
Ala/(Leu+Phe)
-1.26
0.15
0.15
Gly/Ile
0.97
1.47
0.81
Selected variables
282
f3
cellulases
f2
cellulases
f3
0
lipases
amylases
proteases
-2
proteases
-4
-6
amylases
-10
-8 -6 -4 -2 0
lipases
-8
-10
8 10
10
f2
f1
C
10
f2
cellulases
lipases
0
proteases
amylases
4 2
0 -2
-4 -6
f
3
10
0
f1
Fig. VII.7. Score plots on the planes of the first and second (A), and
second and third discriminant functions (B), and on an oblique plane of the
3D space defined by the three discriminant functions (C) of the LDA
model of Table VII.4.
283
Finally, according to Fig. VII.7, part B, the lipase class was resolved with
respect to the other classes (protease+cellulase+amylase) mainly along
discriminant function f3. As illustrated in Fig. VII.7, part C by using a plane
oblique to the three discriminant functions, all the possible pairs of classes were
very well resolved.
Then, the peak areas of the most stable amino acids (Ala, Arg, Asp, Glu,
Gly, His, Leu, Lys and Phe) were exclusively used as predictors to construct an
LDA model. In this case, the following four peak area ratios were selected by the
stepwise algorithm: Glu/His, Glu/Ala, His/Ala, Ala/(Leu+Phe). This model gave
W = 0.387, and the score plots along the discriminant functions showed well
resolved classes. The resolution between classes was only slightly lower than that
observed in Fig. VII.7 for the model obtained with the eight predictors of Table
VII.4.
[Olsen, 1998].
largest single market for enzymes, constituting 25 - 30% of total sales in the
enzyme market
[Novozymes, 2002].
284
extreme temperatures and high pH values, and in the presence of chelate agents
for calcium ions. For this purpose, several techniques (i.e. DNA technology) are
used [Olsen, 1998; Novozymes, 2002].
Enzymes are used in small amounts (0.4 - 0.8% crude enzyme by weight)
in most formulations [Novozymes, 2002], being by far proteases, lipases, amylases
and cellulases the most commonly used enzyme classes. Proteases are used to
remove protein-rich stains (including blood and grass), amylases are addressed to
solubilize starch-containing stains, also preventing starch from adhering to
fabrics and dishes, lipases help in removing fat and edible oil stains, and
cellulases are used to remove fuzz and little balls of fibres from the surface of
cotton fabrics.
Analytical methods for enzyme identification and quantitation in raw
materials and manufactured products are required in industrial quality control.
Further, enzyme producers and their customers are also interested in
investigating the enzyme market trends, which also requires analytical support.
Finally, analytical methods for enzymes are also needed to assess their potential
impact on water treatment plants, as well as to optimize plant operation.
However, in spite of their interest in the detergent industry, and in environmental
and toxicological studies
[www.heraproject.com],
the protein into its constitutive amino acids followed by derivatization and
separation/detection by GC [Gimeno-Adelantado, 2002; De la Cruz-Caizares, 2004;
Rampazzi, 2002]
or HPLC
285
been also used to predict the enzyme class [Beneito-Cambra, 2008; Beneito-Cambra,
2009-B];
however, much information about the nature of the protein is lost during
total hydrolysis.
In proteomics, a common approach for protein analysis is the enzymatic
digestion with trypsin. In this way, the resulting peptides, which are much more
specific for protein identification than the amino acid profiles can be studied.
After digestion, profiles are studied by HPLC-MS
Opiteck, 1997-B; Opiteck, 1998].
packed
[Cunliffe, 2007;
286
peptides
proteins were as follows: an isocratic step with 1% ACN for 2 min followed by a
linear gradient up to 75 % ACN in 18 min.
Absorbance
Absorbance
120
80
100
40
50
40
20
0
10
40
20
150
15
20
Time (min)
10
15
20
Time (min)
As shown in Fig. VII.8, most enzymes gave rise to a single predominant peak;
however, a comparison of enzymes of different classes showed a coelution of the
main peaks for the following enzyme class pairs: proteases and cellulases (main
287
peak within the ca. 11-12 min range), and amylases and lipases (main peak
within the ca. 13-14 min range). No improvement in the resolution between
enzyme classes was achieved by modifying the elution conditions. Then, the
chromatograms of the intact enzymes are useful to assess the quality of the
protein for any enzyme class; however, chromatography of the intact enzymes
did not provide the necessary information to unequivocally identify unknown
enzymes. For this purpose, application of the tryptic digests of the enzymes was
investigated.
Absorbance
120
80
40
0
5
80
10
15
Absorbance
B
40
0
10
20
30
40
50
Time (min)
In addition to the enzymes, BSA was also used as a reference protein to check
the performance of the tryptic digestion, as well as a probe to optimize the
288
Absorbance
10
20
30
40
50
Time (min)
289
290
Absorbance
20
*
C
Absorbance
20
0
0
20
40
Time (min)
20
40
Time (min)
PC = 1 + (t g / w)
(E.VII.1)
where w is the average peak width at 4 (13.4% of peak height) in time units
(experimentally measured) and tg is the gradient time. The global resolution, RG,
was also measured as the geometric mean of the resolution between the
291
consecutive peak pairs. To perform this evaluation unresolved peak pairs or with
RS values 0.5 were excluded.
Fig. VII.12 shows the number of peaks, PC and RG found for a protease
and a lipase using the different columns. As it can be seen for the protease (Fig.
VII.12 part A), the PC values increased in the following order: ProSwift <
Gemini (5 m) < Chromolith ~ Gemini (3 m) < Kinetex, and a similar order
was obtained for the lipase, providing in this case the Chromolith column higher
PC values than the Gemini (3 m) column. A similar trend was observed for the
number of peaks and RG; in both cases, the Kinetex column showed the best
performance.
The differences in number of peaks, PC and RG values among the columns
are a consequence of differences in their morphological features. Comparing
monolithic beds, the results obtained for complex tryptic digests for the ProSwift
column suggest a larger globule/polymer skeleton diameter and/or bed
inhomogeneity than for its silica counterpart (Chromolith Performance RP18),
which translates into lower plate numbers and reduced peak capacities as well as,
along with absence of mesoporous structure, into lower surface area and less
retention of analytes. Silica monoliths show larger porosities than packed beds.
Hence, they exhibit a higher permeability than packed columns. The silica
skeleton in Chromolith is mesoporous and has mean thickness of around 1-3 m
leading to a mass-transfer kinetics faster than that of packed columns with 5-m
particles and comparable to those of columns packed with 34 m particles
[Guiochon, 2007],
The Kinetex column, packed with 2.6 m core-shell particles (porous shell
0.35 m, fused-core 1.9 m) showed a better performance than both Gemini
columns, packed with conventional 5- or 3-m particles and monolithic
supports.This can be explained by the particular characteristics of the Kinetex
292
packing, where the particles are constituted by porous outer layer surrounding a
solid non-porous core [Snyder, 1979].
80
Number of peaks
A
60
40
20
0
Peak Capacity
400
300
200
100
C
20
10
293
Absorbance
40
Absorbance
Absorbance
20
0
40
0
0
20
40
60
Time (min)
[Ruta, 2010].
The
Kinetex column which showed the best chromatographic performance for tryptic
digests, was chosen for further studies. Chromatograms of a protease, an
amylase, a lipase and a cellulase are shown in Figs. VII.11, part D and Figs.
VII.13, parts A to C, respectively.
294
295
Absorbance
40
Absorbance
40
0
0
20
40
60
Time (min)
296
CHAPTER VIII
MONOLITHIC COLUMNS
2005;
2003],
[Svec,
[Peters, 1997; De Vries, 2008; Peters, 1998-A; Yu, 2002; Eeltink, 2005]
and UV irradiation
are the
[Faure, 2007],
[Beiler, 2007]
Mirapeix,
-radiation
2008-A;
Cant-Mirapeix,
electron beam
[Peters, 1997; De Vries, 2008; Peters, 1998-A; Yu, 2002; Eeltink, 2005;
Sfrny, 2005; Beiler, 2007; Cant-Mirapeix, 2008-C; Throckmorton, 2002; DelaunayBertoncini, 2004; Augustin, 2006].
Eeltink,
2005;
Geiser,
2007;
Ngola,
299
2001;
Cant-Mirapeix,
[Peters,
2008-C;
or 2,2-dimethoxy-
2-phenylacetophenone [Augustin, 2008; Ro, 2004; Lee, 2004; Huo, 2007] as initiators.
LPO, as other diacyl peroxides, constitutes a source of free-radicals, when
decomposed by thermolysis, irradiation or activation by tertiary amines [Gu, 2007;
Redington, 1948; Sanchez, 1996; ODriscoll, 1960].
[Gao, 2005].
better permeability and a finer control of the pore size over the studied range of
1,4-butanediol/1-propanol ratio, than columns prepared with AIBN. This
suggested that the combination of LPO and UV irradiation could also be an
attractive way for the fast preparation of LMA-based monoliths.
In this work, the preparation of LMA-based monolithic columns for CEC
by UV irradiation using LPO as an initiator is described. In order to obtain
satisfactory column performances, the composition of polymerization mixture
(i.e. ratios of monomers/porogens and monomer/crosslinker, and the composition
of the porogenic solvent) was optimized. SEM photographs were used to
characterize the morphology of the resulting monoliths, and the CEC
performance of different columns was evaluated by measuring the retention
factors and efficiencies of test mixtures of non-charged solutes. Photopolymerized LMA stationary phases were compared with those prepared by
thermal initiation. These columns were also compared with those prepared using
the more common AIBN instead of LPO as an initiator.
300
with
several
overlappings
(naphthalene/fluorene
and
Absorbance (mAU)
60
40
3
4
20
5+6
7
0
1
4
Time (min)
301
This was
[Gao,
2005].
302
303
Flow rates and retention measured at 5 kV. Mobile phase, 80:20 v/v ACN:5 mM Tris (pH = 8.0)
Weight percentages.
Table VIII.1. Composition of the polymerization mixtures used for the preparation of photo-polymerized LMA-based monolithic
On the other hand, the similar u-values suggested similar flow-through pore sizes
for columns C1 and C2, which was consistent with their SEM pictures (data not
shown). The similarity of the pore sizes could be due to the excessively large 1,4butanediol content employed in the preparation of these two monoliths.
(column
C7).
Other
details
about
the
304
were observed. This trend was consistent with the SEM pictures of these
monoliths. As a representative example, monoliths photo-polymerized with 10
wt% 1,4-butanediol showed a significant decrease in both flow-through pore and
globule sizes when the ratio of monomers/porogens was increased from 30:70 to
50:50 (columns C3C5) (Fig. VIII.2, parts A to C).
Hmin (m)
20
10
0
0
0.5
1.5
60
Hmin (m)
B
40
20
0
0
0.5
u (mm s-1)
1.5
305
[Gao, 2005].
The
C8
compared
with
columns
306
C1,
C4,
C7).
At
50:50
wt/wt
60:40 and 70:30 wt/wt (columns C9, C4 and C10, respectively). When 40:60 was
used the columns exhibited a large flow resistance, which caused column
blockage.
307
4
20
6
10
0
0
Absorbance (mAU)
40
8
B
30
3
20
5 6
10
0
0
12
16
Absorbance (mAU)
60
4
40
1
3
20
5 6
0
0
8
Time (min)
1,4-butanediol/1-propanol
(column
C4);
(B)
50:50
308
When LMA:EDMA ratio was increased from 50:50 (column C9) to 60:40
(column C4), a decrease in retention without any separation improvement was
observed (see Table VIII.1). Finally, with 70:30 (column C10), overlapping of
the naphthalene/fluorene and pyrene/benz[a]anthracene peak pairs was produced.
This was consistent with the SEM pictures of these columns, which showed how
the decrease of the crosslinker concentration from 60 to 30 wt% led to monolithic
beds with larger globules (pictures not shown). This was in agreement with other
studies reported in the literature, where a decrease in the crosslinker percentage
was also accompanied by an increase in the average pore size [Svec, 1995-A; Svec,
1995-B; Okay, 2000].
LMA/EDMA ratio was progressively increased from 50:50 to 70:30 wt/wt, the
hydrophobicity of the monomer mixture was increased, which resulted in an
earlier phase separation. The aggregates preferentially tended to swell with the
LMA monomers than with porogenic solvent. Overall, the globules and voids
formed in this system will be larger. It should translate in a reduction both in kvalues and efficiency along this series. However, similar Hmin values were
observed from column C9 to C4, whereas as expected an increase in Hmin was
observed for column C10. As shown in Table VIII.1, the LMA:EDMA ratio of
60:40 wt/wt (column C4) provided the best Hmin values for all the PAHs of the
test mixture, thus being this ratio selected for the studies that followed. These
efficiencies are comparable with the performance of microparticulate columns
packed with 5 m silica particles [Vlakh, 2007; Eeltink, 2004].
Using LPO, the photo-polymerized LMA-based monolithic columns were
also compared with those prepared by thermal polymerization. For this purpose,
the 1,4-butanediol and porogenic solvent contents, which were found to be
optimal for each polymerization processes were used. The monoliths of the two
types showed similar efficiencies for naphthalene, i.e. Hmin
309
naphthalene
= 11.1 and
[Buszewski, 2004].
comparable with the values of 513 m for PAH compounds (flow rates: 1.01.5
mm/s) obtained with photo-initiated LA monoliths with AIBN
[Geiser, 2007].
However, the plate heights achieved were slightly higher than those obtained for
LA columns chemically initiated with LPO
[Buszewski, 2004],
values for PAH compounds ranged from 2.6 to 5.3 m (flow rates: 0.51.5
mm/s).
Table VIII.2. Reproducibility of CEC properties of LMA-based monolithic
columns photo-polymerized with LPOa
Column-to-column (n=3)
Parameter
Batch-to-Batch (n=3)
Mean
RSD (%)
Mean
RSD (%)
u (mm/s)
1.25
4.5
1.29
5.4
kanthracene
1.08
2.8
1.00
6.3
Hminb (m)
11.0
2.1
13.1
6.0
310
311
a)
Absorbance (mAU)
80
40
3
6
2
0
2
10
Time (min)
As shown in Tables VIII.1 and VIII.3 for LPO and AIBN, respectively,
at the optimal ratios of monomers/porogens (40:60 wt/wt) and LMA/EDMA
312
[Cant-Mirapeix, 2009-B].
shows the separation of five basic compounds in less than 12 min with column
efficiency of up to 20 m, whereas the column initiated with AIBN (Fig. VIII.7,
part B) as in Fig. VIII.6 showed longer analysis times with theoretical plates ca.
24 m. Similar efficiencies were achieved for RP columns, in particular XTerra
RP18 and XBridge C18, two columns with low-silanophilic activity
2003],
[McCalley,
313
can be observed that symmetric peaks were obtained in CEC due to suppressed
electrostatic interaction between the neutral solutes and the positively charged
monolithic surface, then; these stationary phases were suitable for the analysis of
basic compounds.
3
Absorbance (mAU)
0
Absorbance (mAU)
3
2
7
5
1
1
0
0
5
6
Time (min)
314
Absorbance (mAU)
1
4
3
2
Absorbance (mAU)
10
20
30
4
1
0
0
10
20
30
40
Time (min)
315
Organic
photoinduced
Mirapeix, 2009-A]
[Geiser,
2007; Ngola, 2001; Delaunay-Bertoncini, 2004; Faure, 2007; Yu, 2002; Augustin, 2006],
316
other variables such as type of free-radical initiator and its concentration, which
both affect the kinetics of the free-radical polymerization. Consequently, these
will affect the morphology of the resulting polymer.
Most literature concerning UV-initiated polymeric-based monoliths has
reported the use of AIBN
Faure, 2007; Yu, 2002; Augustin, 2006; Augustin, 2008; Bernab-Zafn, 2009]
or
DMPA [Ro, 2004; Lee, 2004; Huo, 2007; Gu, 2007] as initiators, whereas others, such
as BPO and LPO
wt% EDMA and 0.3 wt% META) and 60 wt% porogens (10 wt% 1,4-butanediol
317
and 90 wt% 1-propanol). The AIBN percentage was varied from 0.05 to 0.8 wt%
in the polymerization mixture. As it can be seen in Table VIII.4, minor changes
in efficiency (minimum theoretical plate, Hmin obtained from Van Deemter plots)
were observed for the range of AIBN tested. On the other hand, small variations
in u-values were observed with increasing AIBN content, while the k-values
increased from 0.05 to 0.30 wt% AIBN, followed by a decrease at contents
higher than 0.6 wt% (see Table VIII.4).
N
N
OCH3
N2
OCH3
OCH3
OCH3
OCH3
C OCH3
CH3
OCH3
O
O
O
CH3(CH2)9CH2
CH2(CH2)9CH3
CH3(CH2)9CH2
Fig. VIII.8. Photolytic cleavage schemes of four investigated photoinitiators: AIBN (A), DMPA (B), BPO (C) and LPO (D).
318
319
320
Flow rate and retention measured at 5 kV. Mobile phase, 80:20% (v:v) ACN:water (5mM Tris buffer pH 8.0).
Mean and RSD values (%) obtained for run-to-run repeatability with n = 10 at optimal initiator content.
c
Mean and RSD values (%) obtained for column-to-column repeatability with n = 3 at optimal initiator content.
d
Global resolution as geometrical mean of resolution between the consecutive PAH pairs.
Tabla VIII.4. Electrochromatographic properties of LMA-based monolithic columns prepared with several photo-initiators.
321
Flow rate and retention measured at 5 kV. Mobile phase, 80:20% (v:v) ACN:water (5mM Tris buffer pH 8.0).
Mean and RSD values (%) obtained for run-to-run repeatability with n = 10 at optimal initiator content.
c
Mean and RSD values (%) obtained for column-to-column repeatability with n = 3 at optimal initiator content.
d
Global resolution as geometrical mean of resolution between the consecutive PAH pairs.
It was in agreement with Fig. VIII.9, part B and C, and with the increase in
retention observed from 0.05 to 0.3 wt% AIBN. However, a reduction in kvalues was observed at larger AIBN contents (Table VIII.4). Several possible
explanations could be given for this irregular retention behaviour. The effect of
initiator concentration on the final globule size is likely to be dependent on the
competition between the rates of nucleation and termination. With increasing
AIBN content, an increase in termination step could be favoured, which leads to
the formation of less chains able to grow a sufficient length either to become
nuclei or to stabilize the growing nuclei. It could reduce the available free sites
for interactions within the polymer, and would produce a decrease in retention of
analytes
account the screening effect of initiator, as has been reported [Lai, 1997; Gruber,
1992; Guthrie, 1986; Hutchison, 1973; Moad, 1995].
the distribution of initiating species will become less uniform, being the majority
of these concentrated in a relatively narrow region close to the radiation source.
Thus, this surface layer will effectively screen the deeper layer of initiating
species against UV radiation. This non-uniformity will give rise to an overall
reduction in the rate of polymerization, and consequently, monoliths with less
binding capacity.
The possibility of employing DMPA as free-radicals source by photoinitiation instead of AIBN was also evaluated. Different amounts of DMPA
ranging between 0.02 and 0.2 wt% were tested (Table VIII.4). Concentrations
above 0.15 wt% led to a fast polymerization of mixture at room temperature,
making a proper filling of the capillaries unfeasible. Along the DMPA range,
similar flow rates and Hmin values were obtained, while the k-values decreased
from 0.02 to 0.05 wt% DMPA, remaining practically constant at 0.15 wt%.
However, SEM pictures of these monoliths did not show significant differences
in morphology. A representative example of porous structure of these monoliths
is given in Fig. VIII.9, part D.
322
[Rohr, 2001]
323
Absorbance (mAU)
80
40
6
20
5 6
0
1
160
Absorbance (mAU)
20
120
80
60
80
40
60
60
40
80
3
1
6
40
3
5 6
5
20
2
0
0
1
2
Time (min)
Time (min)
324
decrease at 0.8 wt%. The beds initiated with 0.05 (Fig. VIII.11, part A) and 0.6
wt% (Fig. VIII.11, part C) showed globule sizes smaller than those obtained
with 0.3 wt% (Fig. VIII.11, part B), which would explain its lower retention.
The monolith initiated at 0.8 wt% did not show significant changes in
morphology compared to 0.6 wt%.
325
VIII.12, part A). On the other hand, for initiator contents comprising between
0.050.3 wt% BPO and 0.150.6 wt% LPO, the k-values obtained with BPO
initiated monoliths were lower than those observed for LPO ones. This result was
consistent with SEM pictures, which showed larger globules in beds made with
BPO than those prepared with LPO (see Figs. VIII.11, part B and VIII.12, part
B). However, for BPO contents above 0.6 wt%, the use of this initiator provided
again monoliths with larger retention. As we commented above, these variations
in retention could be probably ascribed to the yield of radical generation, which
depends on the type and concentration of photo-initiator [Gruber, 1992].
326
As observed for LPO, similar k-values were obtained within the range
0.150.8 wt%, in contrast to their BPO counterparts. This fact would allow the
possibility of a fine control of morphological and electrochromatographic
properties of LMA-based monolithic columns prepared using LPO.
In terms of efficiency and resolution, similar values were achieved both
using BPO and LPO over the tested initiator range, except at 0.3 wt% BPO,
where the lowest efficiency and resolution values (Table VIII.4) were achieved,
probably due to its large globule size. Fig. VIII.10, parts C and D shows the
electrochromatograms of PAHs that provided the best chromatographic
performance obtained with the monoliths initiated with BPO and LPO,
respectively.
Finally, a comparison of four investigated photo-initiators was performed
taking into account the optimal initiator concentration found for each one. As
shown in Table VIII.4, the four initiating systems investigated showed in their
optimum conditions similar Hmin and RG values. Fig. VIII.13, parts A to D
shows the Van Deemter plots for several PAHs with the optimal monoliths found
for each photoinitiator. The AIBN and LPO monoliths showed low mass transfer
contributions (C-term): 5.612.1 and 6.812.5 ms, respectively, followed by
DMPA (8.214.3 ms) and BPO (7.816.6 ms). At the sight of these values and
Fig. VIII.13, the AIBN monoliths showed the flatter profile, which offered the
possibility of increasing the speed of analysis without a significant loss in
efficiency. Anyway, as shown in Fig. VIII.10, for all tested monoliths, the PAHs
were satisfactorily separated at 25 kV in less than 4 min. The monoliths initiated
with LPO gave the best compromise between separation performance and
analysis time, followed by AIBN, DMPA and BPO.
The repeatability of LMA-based monolithic columns prepared using the
optimal monolithic columns found for each photoinitiator was also examined
327
Hmin (m)
30
20
20
10
10
0
0
0.4
0.8
1.2
1.6
30
Hmin (m)
30
0.4
0.8
1.2
30
20
20
10
10
1.6
0
0
0.4
0.8
1.2
1.6
u (mm s-1)
0.5
1.5
Fig. VIII.13. Van Deemter plots of LMA monolithic columns photopolymerized with 0.3 wt% AIBN (A), 0.05 wt% DMPA (B), 0.05 wt%
BPO and 0.3 wt% LPO (D). Compounds: () naphthalene, () anthracene,
and () benzo[k]fluoranthene. Polymerization conditions as in Fig. VIII.9
and CEC conditions as in Fig. VIII.10.
328
2
u (mm s-1)
CAPTULO IX
CONCLUSIONES GENERALES /
GENERAL CONCLUSIONS
IX.1. Surfactantes
IX.1.1. APGs
The mass spectra of the APGs in the positive-ion mode were constituted by
the single peaks of the [M+Na] + ions. Using an alkylamide column, isocratic
elution and MS detection, the alkylmonoglycosides can be separated with
resolution between the - and -epimers and ring isomers. The isomers of the
331
Main points:
-
Using a cyanopropyl column and gradient elution, the AMG ring isomers are
resolved in shorter analysis times than using the alkylamide column.
In ESI-MS, the AMG response factors are independent of the content of ACN
in the mobile phase, increase from C1G1 to C10G1, and decrease for longer
alkyl chains.
332
333
In the presence of Na+, the PI MS spectra of D-glucose and the APGs with
alkyl chains up to 12 carbon atoms gave predominantly [M+Na]+ ions. The MS2
spectra obtained from the isolated [M+Na]+ parent ions showed a common
fragmentation pattern with the following features: (i) an m/z 185 ion, which is
due to dehydration and to loss of the alkyl chain as an alcohol in the cases of Dglucose and the AGPs, respectively; (ii) an m/z 143 ion produced by a 0,2A crossring cleavage; (iii) an m/z 413 ion, not containing carbon atoms and probably
containing Na+, OH-, and H2O; (iv) adducts containing a molecule of either Dglucose or the corresponding AGP, M, with the structures [M+413]+, [M+413
2H2O] +, and [M+413+H2ONaOH] +; and (v) a few additional ions, whose
structures depended on the length of the alkyl chain. In addition, D-glucose also
showed a weak m/z 113 ion produced by a
0,3
2,5
A cross-ring
cleavage.
Standards of AMGf were not available; however, the fragmentation
patterns of these ring isomers were studied by using HPLC-MS and HPLC-MS2
of industrial mixtures of APGs. These mixtures contain minor concentrations of
octyl- and decyl-MGf. In comparison with the AMGf, the m/z 413 ion and its
adducts with unfragmented molecules were more easily formed by the AMGp. In
contrast, the m/z 143 ion occurred more easily in the AMGf than in the AMGp,
which can be due to the lower stability of the five-membered furanoside rings.
Main points:
-
In the presence of Na+, the [M+Na] + peak also predominates in the ESI-MS
spectrum of D-glucose in PI mode.
When obtained from [M+Na] + parent ions, the ESI-MS2 spectra of D-glucose
and the APGs show a common fragmentation pattern.
334
IX.1.2. Alcoholes
335
336
[Bernab-Zafn, 2006].
337
338
[Bernab-Zafn, 2006].
339
carboxylic acids are obtained when these acids are initially present in the
sample. In this case, an additional spectrum or chromatogram, obtained without
adding Jones reagent to the sample, can be used to subtract the signal
contributions arising from the carboxylic acids initially present in the sample
and thus to isolate the contributions of alcohols. Although signal subtraction
increases the random error, the loss of precision is small when the alcohol
concentration in the samples is larger than that of the corresponding carboxylic
acids.
Main points:
-
Aliphatic primary alcohols are not detected in MS; however, using this
procedure, the identification and quantification of aliphatic primary alcohols
by ESI-MS is possible.
The oxidation yield decreases with increasing water content in the sample.
340
Ethoxylated alcohols are oxidized with lower yields (60-65%) than those
obtained with non-ethoxylated alcohols (~ 100%), however, oxidation yields
are independent from the number of carbon atoms and the number of EOs.
The proposed method allows the use of evaporative detectors and mass
spectrometry detection in the determination of aliphatic primary alcohols.
The method is direct, fast and results in lower LODs with complex samples of
industrial and environmental origin.
IX.1.3. AES
Sin embargo, cuando los FAEs se esterifican, los AESs se transesterifican, dando
lugar ambas clases de surfactantes a los mismos derivados. Por lo tanto, en el
caso en que las dos clases de surfactantes estn presentes en una muestra, los
cromatogramas resultantes muestran, en realidad, la suma de FAEs y AESs. Por
lo tanto, en este trabajo se ha desarrollado un procedimiento para la separacin
de ambas clases de surfactantes, empleando SPE con un cartucho de SAX. Tras
la separacin, los oligmeros se derivatizan independientemente y se determinan
por RP-HPLC-UV. La separacin casi cuantitativa de las dos clases de
surfactantes, incluyendo oligmeros hidroflicos e hidrofbicos, se logr
341
also transesterified, leading to the same derivatives as FAE. Thus, if the two
surfactant classes are present in the samples, the resulting chromatograms show
the sum of both FAE and AES oligomers. In this work, a procedure for the SAX
342
Main points:
-
Reacting with a cyclic anhydride, FAEs are esterified, and AESs are
transesterified, quantitatively giving rise to the same derivatives (hemiesters
of the alcohol residues).
343
The method allows the use of FAEs as calibration standards for AESs.
IX.2.1. PVP-NO
[Cottet, 2001;
344
indican una fuerte interaccin del PVP-NO con las micelas de SDS, y son
coherentes con la formacin de micelas mixtas de dos tipos: las constituidas por
polmeros libres y SDS, y las que contienen agregados de polmero y SDS
1971;
Cabane,
1977].
[Arai,
[Oakes, 2003-C].
Por lo tanto, en el
The FSCE studies performed in this work have shown that PVP-NO,
which is a nominally non-ionic polymer, behaves as a polylectrolyte, with a welldefined anionic character in aqueous solutions. In this concern, the results
obtained by FSCE agreed with those previously reported using viscosity, light
scattering, conductivity and acid-base studies
Yamamoto, 1996].
The results are also consistent with the presence of at least two
forms of the polymer in aqueous solutions, likely one of them constituted by free
chains (unimers) and the other one by aggregates. This would agree with the CE
studies performed with other polymers and copolymers
1998; tpnek, 2001].
a strong interaction of PVP-NO with the SDS micelles, and are consistent with
the formation of both free polymer/SDS and aggregated polymer/SDS mixed
micelles [Arai, 1971; Cabane, 1977]. This explanation is consistent with the UV-Vis
spectroscopic studies by Oakes et al.
[Oakes, 2003-C].
345
Main points:
-
The FSCE experiments are consistent with the presence of two forms of the
polymer in solution, one made up of free chains (unimers) and the other
constituted by aggregates.
PVP-NO interacts strongly with SDS micelles, giving rise to mixed micelles of
two types: free polymers bound to SDS, and polymer aggregates also bound
to SDS.
346
IX.2.2. PVP
el
347
348
Main points:
-
Working at molar ratios below the saturation point, the stoichiometry of the
complexes (number of monomers per dye ion) can be estimated.
Working in an excess dye (q < 4), the band of the PVP-CR complexes
contains information which is useful to predict the PVP concentration and
their average MW.
349
IX.2.3. PVA
350
complejos PVA-CR, las reas relativas de la banda del complejo y del pico del
colorante libre, la movilidad electrofortica y la constante de disociacin de
pseudo-primer orden del complejo, tambin resultan estar correlacionadas con la
MW y la tacticidad del PVA. Las muestras ms sindiotcticas (tipo H) dieron
complejos con menor movilidad absoluta que las muestras ms atcticas (tipos M
y L). Este comportamiento podra deberse a las mayores distancias entre los
grupos OH adyacentes en las muestras tipo H, las cuales presentan una mayor
relacin rr/mm que las muestras M y L. El producto [qsat + (VD / Vm)]
disminuy para todas las MW, lo que indica que no llega a alcanzarse el rgimen
de drenaje libre (donde la movilidad es constante porque la relacin carga/masa
ya no aumenta al seguir creciendo la MW del polmero). La variacin de este
producto, tambin confirm la dependencia entre la movilidad del complejo
PVA-CR y la tacticidad. Por lo tanto, se puede obtener informacin til acerca de
la MW y la tacticidad del PVA a partir de la CZE de mezclas de PVA-CR. Sin
embargo, sera necesario recurrir a la calibracin multivariante, incluyendo la
variacin ortogonal de las variables MW y el cociente rr/mm, para construir el
correspondiente conjunto de estndares de calibracin. Sin una calibracin
independiente de estas variables no es posible construir modelos capaces de
hacer predicciones de la MW y la tacticidad con una precisin razonable. El
conjunto de muestras comerciales de PVA recopilado y empleado en este trabajo
fue suficiente para demostrar la dependencia de los parmetros de CZE obtenidos
en condiciones de NECEEM sobre la MW y tacticidad, pero no es lo
suficientemente bueno para hacer una calibracin multivariante. Por ltimo,
aunque esto tambin debe ser estudiado, la NECEEM podra ser til para evaluar
la MW y la tacticidad de otros polmeros solubles no cargados.
351
352
samples. Product [qsat + (VD / Vm)] decreased at all MW, thus indicating that a
free draining regime was not reached. The variation of this product also
confirmed the dependence of the mobility per complexed dye ion on tacticity.
Therefore, useful information about both MW and tacticity of PVA can be gained
by CZE of PVACR mixtures. However, multivariate calibration, including the
orthogonal variation of the molecular mass and rr/mm ratio values along the set
of standards, would be necessary to construct multivariate models capable of
making predictions of these two responses with reasonable precision. The set of
commercial PVA samples we collected and used in this work was adequate to
demonstrate the dependence of CZE parameters obtained in NECEEM
conditions on molecular mass and tacticity, but it was deficient to support
multivariate calibration. Finally, although this should be also investigated,
NECEEM could be useful to evaluate MW and tacticity of other soluble noncharged polymers.
Main points:
-
Increasing the PVA concentration up to the saturation point, the band area of
the complex increases linearly, while the sum of the areas due to excess dye
and the dye released during migration, also decreases linearly.
353
The maximum stoichiometry of the complex ranges from qsat ~ 4.9 to 3.5 for
samples of PVA of high and low MW, respectively. This variation occurs at
different values of log MW, depending on the tacticity of PVA.
The shape of the band of PVA-CR complexes, the relative areas of the band of
the complex and free dye peak, the electrophoretic mobility and the
dissociation constant of the complex, are all correlated with MW and tacticity.
IX.3. Enzimas
IX.3.1. CZE
354
It has been shown that CZE of the intact proteins using either basic or
acid BGEs is potentially useful to identify the enzyme brands commonly used in
the formulation of cleaning products. In particular, the basic BGE gives flat
baselines, which makes possible to distinguish a number of small peaks
constituting rather characteristic patterns. In addition to the migration time of
the main peak, the presence of a second large well resolved peak was also a
rather characteristic feature of some raw enzymes. Further, after quantitation by
the Bradfords method, relative sensitivities calculated from the total peak area
of the electropherograms obtained in the basic and acid BGEs can be also useful
to identify the enzymes. Application of the procedure to real samples is possible
in the presence of non-ionic surfactants, but not in samples containing large
concentrations of anionic surfactants, more research being needed to reduce this
interference.
Main points:
-
355
A basic BGE provides a flat baseline on which some small peaks are
distinguished; the peaks constitute characteristic patterns which can be useful
to classify or identify the enzymes.
Relative sensitivities in the acid and basic BGEs, calculated after protein
quantification by the Bradfords method, may also be useful for enzyme
identification.
356
Main points:
-
Sample matrix effects were reduced by including spiked samples in the LDA
training set.
357
358
Main points:
-
IX.3.4.1. Comparacin de columnas microparticuladas y monolticas para RPLC de digestos trpticos de enzimas industriales en productos de
limpieza
Se ha realizado un estudio comparativo de las prestaciones de diversos
soportes cromatogrficos comerciales, incluyendo tanto columnas monolticas
(una polimrica y otra de base slice) como columnas particuladas (dos basadas
en tecnologa convencional de partculas y una conteniendo partculas de ncleo
fundido), para el anlisis mediante HPLC-UV de digestos trpticos de las
enzimas ms comunes en la industria de los detergentes. Se desarroll tambin
un procedimento para el control de calidad de las enzimas intactas en
concentrados industriales, utilizando para ello la columna monoltica polimrica.
Sin embargo, esta columna proporcion unas bajas eficacias y resolucin global
en la separacin de los pptidos obtenidos por digestin con tripsina. Para este
fin, la columna Kinetex, empaquetada con partculas de 2,6 m, con un ncleo
fundido no poroso y una superficie porosa (tecnologa de partculas shell-core),
359
The
chromatographic
performance
of
several
commercial
360
Kinetex column in the optimal elution conditions was applied to the analysis of
tryptic digests from both raw industrial concentrates of the enzymes as well as to
enzymes in spiked detergent bases and commercial cleaners. The resulting
chromatograms were quite similar to those obtained by directly digesting the
industrial enzyme concentrates; then matrix interferences were not observed.
Therefore, HPLC of intact enzymes is useful in the quality control of
industrial samples, as well as to discard enzyme classes; however, a more
selective approach is required for a reliable classification and identification of
the enzyme. For this purpose, HPLC of the tryptic digest of the enzyme using a
core-shell particulate column is an excellent option. Further work on this topic,
in order to establish characteristic peptides leading to the reliable identification
of the individual enzymes by coupling LC with MS is in progress.
Main points:
-
361
362
evalu el potencial de las columnas obtenidas con ambos iniciadores para separar
compuestos tanto neutros como bsicos. Teniendo en cuenta los resultados
obtenidos, el empleo de LPO y radiacin UV es la mejor opcin para la
preparacin
rpida
de
monolitos
de
LMA
con
buenas
prestaciones
cromatogrficas.
Main points:
-
363
364
described. The influence of each type of initiator and its concentration in the
polymerization mixture was studied. As a result of this study, we have found that
the type and variation of initiator content can produce changes in the monolithic
structure, leading to an increase, a small influence, or a decrease on the final
globule size, and therefore, variations in the electrochromatographic properties
of monoliths. Consequently, it is important to find an optimum concentration for
each photo-initiator investigated. Thus, under their respective optimum
conditions, similar efficiencies and resolutions were obtained for the four
investigated photo-initiators. The monoliths initiated with AIBN and DMPA
showed slightly better separations than BPO and LPO, however, this latter
initiator provided shorter analysis time jointly with a fine control in the retention
properties. Additionally, the highest RSD values found for column-to-column
repeatabilities were obtained for beds photo-initiated with BPO.
Main points:
-
The type and concentration of the initiator may cause changes in the
structure of the monoliths obtained by photopolymerization, which implies
variations in the CEC properties.
The four photoinitiators which have been studied show similar efficiencies
and resolutions. The monoliths polymerized with AIBN and DMPA showed
separations slightly better than those polymerized with BPO and LPO;
however, LPO provided the shortest analysis time for the test compounds. On
the contrary, BPO provided the worst column to column reproducibility.
365
CAPTULO X
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ABREVIATURAS
A
AB
ABS
ACN
Acetonitrilo / Acetonitrile
AES
AIBN
,-azobisisobutironitrilo / ,-azobisisobutyronitrile
Ala
Alanina / Alanine
Alc
Alcalase 2.5 L
AMG
Alquilmonoglucsido / Alkylmonoglucoside
AMGf
Alquilmonoglucofuransido / Alkylmonoglucofuranoside
AMGp
Alquilmonoglucopiransido / Alkylmonoglycopyranoside
ANN
ANOVA
AOS
APCI
APE
APES
APG
Alquilpoliglucsidos / Alkylpolyglucosides
APPI
Arg
Arginina / Arginine
AS
Asn
Asparagina / Asparagine
399
Asp
B
BGE
BPO
BSA
C
Car
Carezyme 4500L
CE
CEC
Electrocromatografa capilar /
Capillary electrochromatography
Cel
Celluzyme 0,7T
CGE
CI
CID
CIEF
CMC
CR
Cys
Cistena / Cysteine
CZE
D
DAD
DMPA
2,2-dimetoxi-2-fenilacetofenona /
2,2-dimethoxy-2-phenylacetophenone
400
DMSO
Dimetilsulfxido / Dimethilsulfoxide
DNA
DP
DTI
Dur
E
EDMA
EI
EIC
ELSD
End
Endolase 5000L
EO
EOF
ESI
Esp
Esperase 8.0L
EtOH
Etanol / Ethanol
Eve
F
FAA
FAB
FAE
FID
401
FSCE
FT
G
GC
Gf
Glucofuransido / Glucofuranoside
Gln
Glutamina / Glutamine
Glu
Gly
Glicina / Glycine
Gp
Glucopiransido / Glucopiranoside
H
HAcO
HEC
Hidroxietilcelulosa / Hydroxyethylcellulose
HILIC
His
Histidina / Histidine
HPLC
I
ICP
ID
IDA
Ile
Isoleucina / Isoleucine
IT
402
L
LAS
LC
LDA
LES
Leu
Leucina / Leucine
LMA
LOD
LPO
Ls
Lx
Lipex 100L
Lys
Lisina / Lysine
M
MALDI
MEKC
MeOH
Metanol / Methanol
Met
Metionina / Methionine
META
MGf
Monoglucofuransido / Monoglucofuranoside
MGp
Monoglucopiransido / Monoglucopyranoside
MS
MW
403
N
NaAcO
NAC
N-acetil-cistena / N-acetyl-cysteine
NECEEM
NH4AcO
NI
NMR
NP
NPE
O
OD
OPA
o-Ftaldialdehdo / o-Phthaldialdehyde
OPE
P
PAH
PEA
o-fosfoetanolamina / o-phosphoethanolamina
PEEK
PEG
Polietilenglicol / Polyethylenglycol
Phe
Fenilalanina / Phenylalanine
PI
Pro
Prolina / Proline
PVA
PVAcO
PVP
PVP-NO
Poli(1-xido-4-vinilpiridina) / Poly(4-vinylpyridine-1-oxide)
PVP-PVI
Poli(1-vinilpirrolidona-co-1-vinilimidazol) /
Poli(1-vinylpyrrolidona-co-1-vinylimidazole)
Q
QDA
QTOF
R
RG
RP
RSD
S
Sav
SAX
SDS
SEC
SEM
Ser
Serina / Serine
SFC
SIM
405
SIMS
SPE
Sta
Stainzyme 12L
T
TAG
Triacilgliceroles / Triacilgliceride
Ter
TFA
Thr
Treonina / Threonine
TIC
TLC
TMBA
cido 3,4,5-trimetoxibenzoico /
3,4,5-trimethoxibenzoic acid
TMS
Trimetilsilano / Trimethylsilane
Tris
Tris(hidroximetil)aminoetano /
Tris(hydroxymethyl)amino ethane
Trp
Triptfano / Tryptophan
Tyr
Tirosina / Tyrosine
U
UV
Ultravioleta / Ultraviolet
V
Val
Valina / Valine
Vis
Visible / Visible
406
ANEXO I
A nexo I
A3
A4
A nexo I
A5
A6
A nexo I
A7
A8
A nexo I
A9
ANEXO II
A nexo II
A13
A14
A nexo II
A15
A16
A nexo II
A17
A18
A nexo II
A19
A20
A nexo II
A21
A22
A nexo II
A23
A24
A nexo II
A25
A26
A nexo II
A27
ANEXO III
A nexo III
A31
A32
A nexo III
A33
A34
A nexo III
A35
A36
A nexo III
A37
A38
ANEXO IV
A nexo IV
A41
A42
A nexo IV
A43
A44
A nexo IV
A45
A46
A nexo IV
A47
A48
ANEXO V
A nexo V
A51
A52
A nexo V
A53
A54
A nexo V
A55
A56
A nexo V
A57
A58
ANEXO VI
A nexo V I
A61
A62
A nexo V I
A63
A64
A nexo V I
A65
A66
A nexo V I
A67
A68
ANEXO VII
A nexo V II
A71
A72
A nexo V II
A73
A74
A nexo V II
A75
A76
ANEXO VIII
A nexo V III
A79
A80
A nexo V III
A81
A82
A nexo V III
A83
ANEXO IX
A nexo IX
A87
A88
A nexo IX
A89
A90
A nexo IX
A91
A92
A nexo IX
A93
A94
A nexo IX
A95
ANEXO X
A nexo X
A99
A100
A nexo X
A101
A102
A nexo X
A103
A104
A nexo X
A105