Analysis of neural lipids from microalgae by HPLC ...

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Analysis of neural lipids from microalgae by HPLC- ELSD and APCI-MS/MS . F. Donot, G. Cazals, Z. Gunata, D. Egron, J. Maligne, C. Strub, A. Fontana, S. Schorr-Galindo Francia, 2013 Maestría en Ciencias: Productos Naturales y Alimentos Análisis Químico Cuantitativo Joseoziel Llaguno Guilberto

Transcript of Analysis of neural lipids from microalgae by HPLC ...

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Analysis of neural lipids from microalgae by HPLC-ELSD and APCI-MS/MS.F. Donot, G. Cazals, Z. Gunata, D. Egron, J. Maligne, C. Strub, A. Fontana, S. Schorr-Galindo

Francia, 2013

M a e s t r í a e n C i e n c i a s : P r o d u c t o s N a t u r a l e s y A l i m e n t o s

Análisis Químico Cuantitativo

Joseoziel Llaguno Guilberto

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Introducción

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Microalgas

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Chlorela Vulgaris B. Braunii

1-70 % de lípidos por gramo DW TAG y HC, 67 % gasolina, 15 % turbosina,

Chlorella Vulgaris, Botryocuccus Braunii… (20 % lípidos g/DW) 15 % diesel 3 % residuos

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Fase reversa Fase normal

Amplia separaciónde lípidos

X separación de cada clase

HPLCELSD MS

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Metodología

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Fase orgánica

Extracción soxhlet1 g de biomasa liofilizada

Cloroformo/metanol2:1 v/v

3 ocasiones

ARotavapor

40 ºC13 mbar

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A

1 mL Cloroformo/metanol2:1 v/v

SPE fase normalEPH (extractable petroleum hidrocarbons)

Fase normal

Evaporar extractos Disolver con 1 mL

Acetona

Hexano

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residuo

HPLC-ELSDHPLC-MS-APCI

fase normalAcetona/hexano

Lipidos

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HPLC-ELSDCROMATOGRAFÍA DE LÍQUIDOS DE ALTA RESOLUCIÓN,

DETECTOR DE DISPERSIÓN DE LUZ EVAPORATIVA

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SCL 6BControlador

Shimadzu

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SIL-10ADvpAutoinyector

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Shimadzu CTO-10.ACVP

Horno

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LC-6A

2 Bombas Shidmatzu

Flujo: 0.1 to 10.0 ml/min

Presión maxima de operación: 6000 psi (400 bar)

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Columna y precolumnaLuna/ Phenomenex

Diámetro interno (id) 4.6 mmLargo (mm) 250 mmModo fase normal (silica gel)Forma de partícula esféricaTamaño de partícula (dp) 5 µm

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Sedex 55, Sedere

Bajas temperaturas

Detector: ELSD

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HPLC-MS-APCICROMATOGRAFÍA DE LÍQUIDOS DE ALTA RESOLUCIÓN,

ESPECTROMETRÍA DE MASAS MODO DE IONIZACIÓN QUÍMICA A PRESIÓN

ATMOSFÉRICA

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HPLC Alliance 2790 waters

Componentes

Sistema de flujo de disolventes

Sistema integral de lavado de émbolos

Horno de la columna

Calentador de la muestra

Válvula de selección de la columna

Teclado

Display LCD

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Columna/Luna Si Phenomenex

Diámetro interno (id) 4.6 mmLargo (mm) 250 mmModo fase normal (silica gel)Forma de partícula esféricaTamaño de partícula (dp) 5 µm

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Detector: Espectrometría de masas

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Filtro de masas de cuadrupolo simpleAnálisis MS m/z 20,000 Celda de collision de hexapolo para análisis MS/MSAnálisis MS/MS selección de ion padre m/z 4000 energía de colisión +/- 205 eV

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Fuente de ionización: APCIatmospheric pressure chemical ionisation

Ionización via a transferencia de protón (ion positivo) o abstracción de protón negativo,La muestra es vaporizada en un nebulizador, posteriormente se lleva a cabo la abstracción o transferencia de protón.

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Condiciones

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Gradiente

20

FFADAG

TAG

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Temperatura columnaHexano 0.1 mL/min8 h

Acondicionamiento

1 mL/min

Flujo

30 ºC

Volumen de inyección

10 µL

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ELSD

Temperatura 70 ºCAire comprimido 2 barGanancia de fotomultiplicador 6

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Temperatura de la columna

Hexano 0.1 mL/min8 h

Acondicionamiento

50-2000 Da

Peso molecular

30 ºC

Volumen de inyección

50 µL

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Detector

4000 VCono 20 V

Temperatura muestra 100 ºC Temperatura ionización 400 ºC

50-2000 Da

Peso molecularVolumen de inyección

50 µL

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Discusión ESTANDARES

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TAG

FFA

DAG

MAG

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TAG

FFA

DAG MAG

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FFA

DAG

MAG

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Discusión MUESTRA HPLC-APCI-MS

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TAG

FFA

DAG

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Conclusiones

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TAG

FFA

DAG

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