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Cromatografía de Líquidos
Retos y Oportunidades
M C José Manuel Morales H
Separación en fase líquida – Portafolio de Productos
1290
1260
Bin
1220
Grad
1220
Iso
SFC 1260 SFC and SFC/MS
1260 Hybrid SFC/UHPLC-MS
Bio-inert LC 1260 Bio-inert LC
1260 Bio-SEC
1290 II
1260
Quat
1260
Iso
Dissolution 708/709, 400, BIO-DIS,
8000, MQ 850
High-end UHPLC
Core UHPLC
HPLC-Chip/MS UHC-NCE, Phospho Peptide
mAB-Glyco, HiP Protein ID
Lowflow LC 1290 Micro, 1260 Cap
1260 Nano
GPC-SEC 1260 GPC, 1260 MDS,
GPC 50, GPC 220
Cap. Electrophoresis 7100 CE and CE/MS
Workflow Automation Solutions Method Dev, ISET, 2D-LC, Online SPE, Multi-method/Walk-up, HT LC/MS, ACR,
Online Sample Prep, HDR-DAD, Mobile LC, Online Sampling, etc
HDR
Preparative LC 1260 uS, 1260 AS, 1260 PS
218, SD1
Sample Collection & Preparation
Separation, Purification Detection, Identification, Quantitation
Analysis & Sharing of Results
Service and Support
1200 Infinity Series Method Development
1200 Infinity Series LC
LC columns
AssayMap Bravo
Off-Gel
LC/QTOF
LC/MS
mAb-Glyco Chip LC/MS MassHunter
BioConfirm
MassHunter Suite
MassHunter Suite
ICP-MS ICP-OES
GC/MS
LC/MS
UV-Vis
1260 Infinity Purification
BioCel
Encore Multispan
Filtration & Clean-up
1260 Bio-inert LC
Bio-Columns 1200 Infinity
Series LC
2100 Bioanlayzer
SFC CE
1200 Infinity Series LC
LC columns
GC
Dissolution 1260 Infinity Purification
Filtration & Clean-up GC Headspace
e.g. The Medicinal Chemistry Department
e.g. The Chemical Entity QC Lab
e.g. The Protein Core Facility
High Throughput Screening and compound management Automation: Biocel, Benchcel, Bravo, Encore, RapidFire
Medicinal Chemistry Purity analysis: LC and walk-up LC/MS Purification: Prep LC Characterization: LC/MS
Early ADME/PK screening RapidFire, LC/QTof (qual/quan) & LC/QQQ MS/MS
1200 infinity series Method development solution
1200 Infinity Series LC
columns Single Quad MS
QTof MS/MS
QQQ MS/MS
Biocel Benchcel
Bravo
RapidFire
1260 Infinity Purification systems
Encore
Drug Discovery Drug Development Manufacturing
Process Development and Formulation Development/CMC Analytical method development & transfer for purity analysis : (U)HPLC Organic Impurity analysis: LC, LC/MS, SFC, CE, NMR Inorganic Impurity analysis: IC-PMS/OES Residual solvent analysis: GC, GC/MS Stability Studies: LC/UV, LC/MS Dissolution Testing
Regulatory PK studies Bioanalysis: LC/QQQ MS/MS, DBS
1200 Infinity Series LC
columns
Single Quad MS QTof MS/MS
QQQ MS/MS
OpenLab CDS
Method development solution
SFC
CE
ICP-MS
ICP-OES
Dissolution
Bond Elut SPE MassHunter software
GC/MS
Drug Discovery Drug Development Manufacturing
Quality Control Raw material analysis: FTIR, LC Purity analysis of end products (API/Formulation): LC, LC/MS Residual solvent analysis: GC/headspace, GC/MS Dissolution Testing Cleaning Validation: FTIR, LC
Compliance CrossLab services (multivendor) ; Analytical Instrument Qualification (AIQ)
GC columns
1200 Infinity Series LC FTIR
Dissolution
GC
Drug Discovery Drug Development Manufacturing
EXPECTATIVA
Resolution optimized
3x100mm, 1.8µm
Resolution peak 5 = 7.16
Run time 7 min
Speed and resolution optimized
3x50mm, 1.8µm
Resolution (peak 5) = 4.79
Run time 1.1min
Conventional
4.6x150, 5µm
Resolution peak 5 = 4.20
Run time 11 min
min 0 2 4 6 8 10
min 0 2 4 6 8 10
min 0 2 4 6 8 10
10x PRODUCTIVIDAD Y MEJORA EN LA CALIDAD DE LOS DATOS
1220 Infinity
1260 Infinity
1290 y 1290II
Infinity
La nueva Serie Agilent Infinity
• Modular
• 1300bar, 240Hz
• 10x Sensitivity
• Bajos costos de mtto
• Method Transfer Techn.
• Modular
• 400bar => 600bar
• 20Hz => 80Hz
• 2-10x UV Sensitivity
• Integrated
• 400bar => 600bar
• 20Hz => 80Hz
Transferencia de Métodos “Correr el método y obtener los mismos
resultados“
? 1200
1100
1220
1260
1290
?
?
Transferencia de Métodos: 1260 Quat / 1220 Grad Compatibilidad 100%
1260 / 1220 Infinity LC
Quat / Gradient system
1100 / 1200 Series
Quaternary system
Column: 4.6 x 150mm, 5µm
Gradient: 0 min 20 % B
10min 95 %
Flow rate: 1 mL/min
min 0 2 4 6 8 10
Corre los métodos de HPLC actuales y obtén los mismo resultados !
Transferencia de Metodos : Fácil
LC Agilent 1290 Infinity Rango de trabajo–hasta 1300 bar
0
200
400
600
800
1000
1200
0 1 2 3 4 5
bar
ml/min
Standard LC
MARCA X
Agilent RRLC MARCA X
MARCA X
MARCA X
Agilent 1290 Infinity II LC
Todas las columnas
analíticas
Trnaferencia de Métodos
Mayor Resolución
Análisis más rápidos
Mayor Sensibilidad (MS)
Solventes más viscosos
Temperaturas más bajas
LC Agilent 1290 Infinity Rango de trabajo–hasta 1300 bar
Mayor oferta de módulos
1260 Infinity LC binario (G1312B pump), cuaternario (G1311B pump) e Isocrático (G1310B pump).
El sistema 1260 Cuaternario puede ser usado para aplicaciones semipreparativas con flujos de hasta 10
mL/min.
Sistema Modular, ventajas para crecimiento o reemplazo de módulos sin alterar el resto del sistema.
Técinas como SFC de fácil acople al sistema.
Bombas: Rango de presiones
• Factor importante, posibilidad de poder elegir la columna ideal y tener los resutlados esperados: HPLC
o UHPLC.
• 1260 Infinity LC - 600 bar (5 ml/min), es el sistema ideal, que permite apicaciones de HPLC y
UHPLC. Columnas de 4.6 mm, 3.0 mm y 2.1 mm también sub-2µm (idealmente en columnas de 4.6 y
3.0mmID ) también para columnas de material poroso.
• Posibilidad de elegir el tipo de bomba adecuada a la aplicación (robustez, precisión, estabilidad, etc.)
• Degasificación en línea.
Inyectores: Mayor precisión en la inyección
Diferentes tipos de automuestreador: manual, viales y and vial/well-plate
Menor carry-over con sistemas específicos (G1367E) y mayor precisión en la inyección, i.e. < 0.25% RSD
Posibilidad de control de temperatura
Mayor capacidad de muestras.
Compartimiento para columnas
Capacidad de control de temperatura
Capacidad para columnas de hasta 30 cm
Sistema de válvulas que dan mayor flexibilidad a laboratorio
Sensibilidad en detectores
• Agilent 1260 Infinity DAD (G4212B) y su celda de flujo de 60 mm con opción de crecimiento a sistema HDR-DAD que brinda un rango lineal 30X mayor que el resto de los sistemas.
• Posibiildad de acoplar una gran variedad de detectores UV-Vis, FLD, RID, ELSD, MSD, especiales como en polímeros o biotecnología.
Control del sistema
• Open LAB CDS
• Workstation
• Redes
Operación
Costos de operación y mantenimiento bajos
Crecimiento de HPLC a UHPLC, gradual
Source: Credit Suisse research survey. Analytical Instruments 2011 Survey.
Qué debo saber para elegir un equipo de
Cromatografía de Líquidos
1.- ¿Cuál es el rango de flujos en los métodos que piensa implementar?, ¿Gradientes?
2.-¿Piensa manejar diferentes volúmenes de inyección?, ¿Control de temperatura?, ¿Cantidad de muestras?
3.- ¿Quiere usar una columna específica para cada método, posibilidad de manejar más de una columna?, ¿Varios métodos?, ¿Control de la temperatura?, ¿Dimensiones de columnas?
4.- Cuál es el nivel de concentración en que se medirán sus muestras?, ¿Qué sensibilidad requiere?
etc
Sistemas de Cromatografía Preparativa de Agilent Technologies
Sistema Modular
1200 Infinity Series
1220 Infinity
Isocratic, manual
Gradient, manual
Gradient, automated
Gradient-DAD
1260 Infinity
Low-flow
Cap.LC
Nano-LC
Chip-LC
Analytical flow
Binary
Quaternary
Isocratic
Binary-SFC
Quaternary Bio-Inert
Prep-flow
1290 Infinity
Binary
Quaternary
new
new
• Plataforma abierta con más de 60 Módulos
• Modular y escalable
• Permite actualización escalonada – también para equipos de
las series 1100/1200!!!!
• Plataforma única en el mercado con la mayor flexibilidad
Preparative LC
1220/1260/1290
Purification
1220
Iso/ Grad
Analytical LC
1260
Iso/ Quat/ Bin
Low Flow LC
1260
Nano
1290
Bin
• Plataforma común de HW, FW, SW
• Mayor ciclo de vida, Robustez,
Confiabilidad
• Alto desempeño
• “El mayor valor para cualquier
presupuesto“
1260
HPLC-Chip
1260
Capillary
Sistemas especiales
Alta productividad Análisis Preparativo
LC Bio-inerte
• Titanium, PEEK y libre de SS
SFC
• Caso especial para Bio-Diesel con detectores de UV-Vis y
FID EN EL MISMO EQUIPO!!!!
LC de Flujos bajos Análisis GPC/SEC
Análisis de AA
Sistemas especiales
Cromatografía 2D
A 1290 Infinity TCC Cluster es el corazón del sistema
Hasta 8 columnas
Hasta 26 disolventes
1300 bar ó 600 bares
Mas de 1000 condiciones de trabajo
programables
4-6 diferentes zonas de temperatura
controlada
Para columnas de 2.1 – 4.6 mm ID y
hasta 300 mm de longitud
1290 Infinity Thermostatted
Column Compartment Cluster
Desarrollo de Métodos Solución Multi-metodo
1290 Infinity\1290
Brochure
espanol.pdf
Sistemas especiales
Aminoácidos
ELSD – Detectores especiales
INULINAS
Aplicaciones en Desarrollo de Métodos
Publication
Number
Title Type Resp.
Agilent
Author
Publication
Date
YYYY/MM/DD
Separation
System
(LC, CE, SFC or
specific
system)
Detection
System
(Mass
Spectrometer/
LC Detector)
Focus
(Featured
product or
specific
functionality)
Stationary
Phase
Market Sub-segment
5991-4732EN
Method Development for Enantiomers Using the Agilent
1200 Infinity Series Method Development SolutionAN
Angelika
Gratzfeld-
Huesgen
2014/06/01 MethDev DAD 8-pos/9-port
valve; Method
Scouting Wizard
N/A Small Molecule
Pharmaceuticals &
Generics
Drug Development
5991-3834EN
Quality-by-Design Approach to Stability Indicating Method
Development for Linagliptin Drug Product
AN
Syed Salman
Lateef
2014/02/01 1290 Bin;
MethDev
DAD 8-pos/9-port
valve; TCC
cluster; Fusion
AE Software
Bonus-RP;
Eclipse Plus C8;
Extend-C18;
PLRP-S 100Å; SB-
C18; SB-Phenyl
Small Molecule
Pharmaceuticals &
Generics
Drug Manufacturing &
Quality Control
5991-3212EN
Optimizing the Separation of 20 Nitro-aromatics Using
Consecutively, a Phenyl-Hexyl Column with p-p Interaction
and a C-18 Column on the Agilent 1290 Infinity Quaternary
Method Development Solution
AN
Angelika
Gratzfeld-
Huesgen
2013/10/01 1290 Quat;
MethDev
DAD 8-pos/9-port
valve; Method
Scouting Wizard;
TCC cluster
Poroshell 120 EC-
C18; Poroshell
120 Phenyl-
Hexyl
Environmental Soil, Sludges &
Sediments
5991-2784EN
Optimizing Analysis of Phthalates in Plastic Toys Using the
Agilent 1290 Infinity Method Development SolutionAN
Angelika
Gratzfeld-
Huesgen
2013/09/01 MethDev; 1290
Quat
DAD TCC cluster; 8-
pos/9-port
valve; Method
Scouting Wizard
Eclipse Plus
Phenyl-Hexyl;
Eclipse Plus C18
Food Testing &
Agriculture
Pesticides,
Mycotoxins & Other
Contaminants
5991-2129EN
Fast Method Development Using the Agilent 1290 Infinity
Quaternary LC System with Column Selection ValveTO
Angelika
Gratzfeld-
Huesgen
2013/04/01 MethDev; 1290
Quat
DAD 6-pos/14-port
valve
SB-C18; Eclipse
Plus C18; Extend-
C18; Eclipse Plus
Phenyl-Hexyl
All N/A
2D-LC
From 1D-HPLC to 2D-LC
Valve applications – any switching
operation for e.g. column or solvent selection
Valve solutions – complete solutions as e.g.
http://www.chem.agilent.com/Library/brochures/
5991-0291EN.pdf http://www.agilent.com/chem/infinity-online-spe incl. video!
1200 Infinity Series Quick-Change Valves
Aspectos generales en las ciencias de la
separación
„Resolving power is what it is all
about in analytical separation
science.“
Bringing in a 2nd Dimension - how?
Pump Autosampler Column Detector
Pump
Detector
Co
lum
n
Valve
(„Sampler“)
1D-LC System
2D-LC System
+
1D-LC System
„Just like 1D-LC with a chemically selective detector“
Taxanes from taxus extract
Where 2D-LC can help
• Taxol® (Paclitaxel) excellent antitumor activity against
breast and lung cancer.
• Content in Taxus brevifolia very low, synthesis very difficult due
to complex structure.
• Monitoring of content in crop cultures highly desired, but
difficult due to complex herbal matrix
Application Note 5991-3576EN.pdf 1D-Chromatograms of standard mix and plant extracts
2D-LC – Heart-cutting vs Comprehensive 2D-LC
Heart-cutting 2D-LC (LC-LC):
LC1
LC2
Parts of the 1D effluent are injected
onto
2D system
2D-LC – Heart-cutting vs Comprehensive 2D-LC
Comprehensive 2D-LC (LCxLC):
LC1
LC2
1st peak from
1st dimension
2nd peak from
1st dimension
3rd peak from
1st dimension
The whole 1D effluent is injected onto
2D system
(Ultra)Short 2D gradients necessary
Good data quality with fast pumps &
detector
Full („comprehensive“) 2D information!
2D-LC – Heart-cutting vs Comprehensive 2D-LC
Comprehensive 2D-LC (LCxLC):
LC1
LC2
1st peak from
1st dimension
2nd peak from
1st dimension
3rd peak from
1st dimension
LC1
LC2
min
sec
1290 Infinity 2D-LC Solution – Scalability
The right solution from medium to highly complex
samples
Heart-cutting 2D-LC
Sa
mp
le c
om
ple
xity
Multiple heart-cutting 2D-LC
Comprehensive 2D-LC
Comprehensive 2D-LC/(IMS)-MS/MS
API impurity
profiling
Complex
formulations
Bio-
pharmaceuticals
Natural products,
biological
samples....
1D-Setup
Switchable 1D/2D Setup
Change between UHPLC and 2D-LC without manual
Interaction
2D-Setup
Technical Note 5991-4843EN
Protein ID & Peptide Mapping Analysis of Monoclonal Antibody Digest
Application Note 5991-2880EN
Analysis of Monoclonal Antibodies Differentiation of stressed and not stressed Antibodies
Application Note 5991-2880EN Application Note 5991-4530EN
2D-LC
Applications
More Application Areas and Opportunities
Quality Analysis of Extra Virgin Olive
Oils
• RPxRP
• Determination of Phenolic Compounds
Separation of Homologous Series of
Technical Detergents
• HILICxRPLC
• ELSD as detector
• Absolutely orthogonal seperation!
Achiral-Chiral Analytic of Pharmaceuticals
• RPLC x chiral LC
• Ideally suited for the analysis of impurities
in chiral pharmaceutical substances
More Application Areas and Opportunities
Grape juice
Antioxidants
juice
Phenolic compounds in Juices
• Identificiation based on
phenole profile
Allergenes in Food • High matrix load
• Complex analyte spectra of
allergenic proteins
Marine Biotoxines • Highyl complex analyte spectra
• Many isomers with same m/z in
Mass Spec
• Isotopes overlay in MS often
no differentation possible
Ovatoxines a-e
Fluidos Supercríticos
(SFC)
Why Are Customers Interested in SFC?
• Replacement for Normal Phase Chromatography
• Allows aqueous samples
• Complementary to RP-LC or HILIC
Unique Selectivity / Orthogonal to LC
• High analysis speed – faster than LC!
• Lower operational costs and a green technology!!!
CO2 Mobile Phase / Very Little Organics
• Broad detector compatibility - UV, MS, ELSD…
• Various instrument configurations
Versatile
• Premier choice for chiral compounds
• Many applications for achiral compounds!
• Polar and non-polar compounds in a single run Chirals and more!
• LC-like robustness & sensitivity combined with SFC speed and orthogonal separation selectivity
Wide application range in many industries
Pestaña de SFC en MassHunter
Solo un punto de ajuste para
SFC:
Back pressure
Dos formas de conectar a un detector de Masas
Split Flow Only a small portion of the effluent goes to the MS
• High Resolution
• Qualitative Analyses
• Screening and Identification
• New MS passive Splitter Kit G4309-68715 - unique technology!
Full flow – Agilent Only All effluent goes through the BPR into the MS
• High Sensitivity
• All molecules make it into the source
• Good for quantitative analyses
• Low dispersion nozzle reduces dead volume (new nozzle in development)
Agilent 1260 Infinity con SFC/FID
• Cumple con ASTM 5186 y
otros métodos
estandarizados para la
determinación de
insaturación en aceites,
diesel y biocombustibles
• Añade FID al sistema SFC
Ambiental - Alimentos
