UPC Sistema per la Convergenza Cromatografica · 2013-07-10 · Advancements in Gas Chromatography...
Transcript of UPC Sistema per la Convergenza Cromatografica · 2013-07-10 · Advancements in Gas Chromatography...
©2013 Waters Corporation – Seminari UPLC-MS 1
UPC2
Sistema per la Convergenza
Cromatografica
Andrea Perissi,
Specialista Applicativo
©2013 Waters Corporation – Seminari UPLC-MS 2
What is a Supercritical Fluid ?
Gas - like low viscosity confers high diffusivity and limits pressure drop
Liquid - like high density ensures good solvent properties
High diffusion coefficient enhances kinetic performance of SFC
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Which Supercritical Fluid ?
Courtesy of A. Grand-Guillaume Perrenoud, D. Guillarme, Pr J-L. Veuthey, University of Geneva
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Why CO2 ?
CO2 reaches supercritical state at 31.1°C and 73.8 bar
– Its physical state can be easily manipulated
CO2 is non toxic, non flammable
CO2 is chemically pure, stable and non-polar solvent, also compatible with LC detectors
Carbon Dioxide as a Green Solvent
– Recovered from industrial and fermentation plants
– Avoids the production of CO2 that would have been generated from disposal of the solvents it replaces.
– Less time and energy are used to evaporate fractions to get to pure analytes.
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Advancements in Gas Chromatography
Gas Chromatography
Since the advent of the use of fused-silica capillaries in the late 1970’s, only small incremental advancements in GC have occurred.
GC Capillary GC
Minimal development in gas chromatography over the past 30 years
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Advancement in Liquid Chromatography
Liquid Chromatography
The advent of UPLC in 2004 has revolutionized liquid chromatography, providing significant advancement in sensitivity, resolution and throughput
LC UltraPerformance LC
[UPLC]
The practical and functional limits of LC technology are starting to be reached
ACQUITY UPLC I-Class System
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Advent of Convergence Chromatography
Convergence Chromatography
UltraPerformance Convergence Chromatography is the result of significant technological advancements in Supercritical Fluid Chromatography that finally enable this technique to become a reliable and robust analytical tool.
SFC UltraPerformance
Convergence Chromatography [UPC2]
Data courtesy of Davy Guillarme, Jean-Luc Veuthey LCAP, University of Geneva, Switzerland
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Separation Technology Overview
Gas Chromatography
Liquid Chromatography
Convergence Chromatography
Technique focused on non-polars and volatiles Separation achieved by a temperature gradients
• Low efficiency (per unit length) [N]
• Limited selectivity [α]
Technique focused on expanding polarity range and non-volatility Separation achieved by a Increasing Efficiency
• Moderate efficiency [N]
• Moderate selectivity [α]
Technique focused on a unified approach Separation achieved by a Manipulating Selectivity High efficiency [N] • High selectivity [α]
GC
LC
CC
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Higher speed and throughput
– Better peak shape
– Better resolution
– Faster re-equilibration
– Shorter cycle time
– More samples per day
SFC complements RP-LC
– Normal phase technique
– MS compatible
– Different selectivity
– Different elution order
– Better retention of polar compounds.
SFC is proven better for separating isomers
and enantiomers, and structurally related
compounds.
Not a total replacement for HPLC, but it
handles ~80% of all small molecules
SFC Advantages
Zhao Y. et al. LC GC Europe, 2004, 174 (4), 224-238
©2013 Waters Corporation – Seminari UPLC-MS 10
Why has SFC not been adopted?
Despite the potential advantages of SFC, it has never been widely accepted...why?
Lack of robustness – Shifting retention times
– Low accuracy for injection volumes
– Unstable modifier delivery at low percentages of co-solvent (< 5%)
Lack of instrument performance – Insufficient instrumentation reliability (pumping
system, injection mechanism, backpressure regulator)
– Large system dispersion and dwell volumes prevented adoption of smaller particles and high throughput analysis
Low sensitivity – High detector and pump noise
– Refractive index effects of CO2
What if those challenges could be
addressed?
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… in the ACQUITY Family
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Gold Award at Pittcon !
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ACQUITY UPC2 System
ACQUITY UPC2 Binary Solvent Manager Pumps liquid CO2 and desired co-solvent (select from up to 4 co-solvents)
ACQUITY UPC2 Sample Manager Fixed-loop injector Perform Partial Loop Injections
ACQUITY UPC2 Convergence Manager •Manages CO2 inlet •Auxiliary inject valve •Active back pressure regulator
ACQUITY UPC2 Column Manager Select from up to 6 columns for method screening
ACQUITY UPC2 Detection •Photodiode array •Evaporative light scattering •Mass spectrometry
Over 20 patent applications!!
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Flow splitter and make-up pump are recommended when co-solvent % is less than 5%
Interfacing ACQUITY UPC2 with MS Detection
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©2013 Waters Corporation – Seminari UPLC-MS 16
ACQUITY UPC² Column Chemistries
First range of sub-2 µm column chemistries
– Designed and tested for the ACQUITY UPC² system
– High efficiency and resolution
– Wide range of selectivity
3.5 µm column chemistries
– Maximum flexibility to tackle any challenging routine analyses
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ACQUITY UPC² Column Chemistries
Available now in sub-2-µm column formats
Available Q2 2012 in 3.5 µm particle size and all HSS columns
Viridis® brand – All 4 chemistries in 5 µm
BEH 2-EP (2-Ethylpyridine) • Good retention, peak shape and selectivity
BEH • Heightened interaction with polar groups such as phosolipids
CSH Fluoro-Phenyl • Good retention of weak bases • Alternate elution orders for acidic and neutral compounds
HSS C18 SB • Analysis of glycerides across vertical markets (Pharmaceutical, Food, Chemical Materials)
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Unified Classification
CO2/MeOH 90/10
Courtesy of C. West, E. Lesellier, ICOA – University of Orléans
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UPC2 Applications by Market
Pharmaceutical Food/Env Chemical Materials Clinical Chiral drugs
Transfer from USP methods
Fat-soluble vitamins
Lipid analysis
Achiral impurity analysis
Chiral pesticides
Explosives
OLEDs
Azo dyes
Non-ionic surfactants
Polymer additives
Drugs of abuse
Vitamin D metabolites
Carotenoids
Positional isomers
Steroids Steroids
Extractables and leachables
Reaction monitoring
Library screening
DMPK/Bioanalysis
Natural products
Metabolomics
Glycerides
PAH
Lubricants
©2013 Waters Corporation – Seminari UPLC-MS 20
Note Applicative
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AU
0.00
0.05
0.10
Minutes
0.0 5.0 10.0AU
0.00
0.02
0.04
0.06
Minutes
0.0 5.0 10.0
Chiral Separation of Permethrin
UPC²
NPLC NPLC SFC
Flow rate
(mL/min) 1 4
Mobile phase hexane:ethanol=90:10 CO2:methanol:DEA=9
5:5:0.2
Back Pressure
(bar) n/a 120
Temperature (C) ambient 40
Column CHIRALCEL OJ-H
(4.6 250 mm, 5 µm)
CHIRALCEL OJ-H
(4.6 150 mm, 5
µm)
Sample Conc. 2 mg/mL
Injection volume
(µl) 10
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Questions ??
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A Novel, Practical Approach to SEC/GPC Analysis
Introducing ACQUITY Advanced Polymer
ChromatographyTM (APC) System
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Introduction of the ACQUITY – APC system
– Launched at Pittcon (Philadelphia) on March 18th
Implementation of sub-2µm /sub-3µm particles
for polymer characterisation (GPC/SEC)
– Based on rigid hybrid particles (BEH chemistry
introduced in 2004)
Complete solution including
– New columns with higher efficiency for both aqueous
and organic size exclusion chromatography
– New chromatographic system specifically designed
for high efficiency columns
o Low dispersion, precise flow rate
– New management of solvent waste and vapor
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GPC:1964 to today
Little/no change in Column technology
Primarily polymer-based resins
– Styrene-DVB
– Methacrylates
Low resolution technique
– Particle size reduction from ~75 micron to ~5 micron
– Instrumentation dispersion limitations
Some attempts for introducing “universal columns” and fast GPC
columns
– Compromises speed for resolution
– GPC/SEC remains a slow technique
The technique of GPC/SEC used in this industry may not have
advanced in 20 years, but the economic, competitive and market
dynamics of the polymer industry have, driving a need for better
information and higher quality data……..faster
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ACQUITY APC System
Analytical Challenges
ACQUITY APCTM System
Gel Based Columns
- Styrene DVB and
methacrylate based
columns are relatively
fragile and typically
cannot easily be
converted from one
solvent to another
Speed of Analysis
- Current approaches to
reduce analysis time
of a GPC assay
compromises peak
resolution and
therefore
characterization data
quality
Lack of Resolution of
Low Molecular Weight
Polymer and
Oligomers
- Traditional GPC
remains to be a low
resolution technique
that is inadequate in
providing the
characterization
information required
for today’s innovative
polymers and building
blocks
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ACQUITY – APC System A Definition
Application technique for the size based separation of
polymers in solution using columns packed with sub-
3µm rigid, high pore volume hybrid particles
combined with a fully optimized low dispersion
ACQUITY system
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Introducing the ACQUITY Advanced Polymer Chromatography (APC) System
Precise
solvent
management
Low system
dispersion
Compatibility
with
challenging
solvents
Rigid, solvent-
resilient
columns
Versatile
column
management
Stable
refractive
index
detection
Flexible
detection
techniques
Wide range of
APC
standards
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New and Innovative Polymers
Polystyrene
Standard 510 Mp Alliance 2695/2414 6x150 HSPgel HR1
ACQUITY APC with RI 4.6x150mm; 45Å XT
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Higher resolution and speed of APC allow for higher number of calibration standards
More calibration points Better calibration Better characterization
FASTER calibration Calibrate in less than 30 minutes, not hours
DAILY calibration, not weekly Better data consistency and quality
GPC APC
28.00 4.90
Speed of Analysis - Better Characterization
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Gel Based Columns vs Rigid Hybrid Particles
THF
DMF
Toluene
One System. One Bank of Columns. Solvent Flexibility.
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Ethylene-Bridged Hybrid (BEH) ACQUITY – APC Columns
More than 10 years of experience with sub-2µm particles
submitted to extreme conditions
– pH, temperature, back pressure, flow rates, …
The BEH particles combine the advantages of both silica and
polymeric material
– Rigid, pressure resistant, easy control of particle size and pore
diameter
– Inert, resistant to temperature changes
E-cord device
– Keep a trace of the usage history (QC certificate & injection count,
temperature, back pressure)
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Rigid Hybrid Columns
82
27
2
µR
IU
-2.00
0.00
2.00
4.00
58
42
2
µR
IU
-2.00
0.00
2.00
4.00
81
70
9
µR
IU
-2.00
0.00
2.00
4.00
81
36
5
µR
IU
-2.00
0.00
2.00
4.00
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50
Poly(methyl methacrylate co ethylacrylate)
THF
Poly(methyl methacrylate co ethylacrylate)
THF
Poly(9,9 di-n-octylfluorenyl 2,7 diyl)
Toluene
Poly(bisphenol-A-co epichlorohydrin) DMF before after % change
Mp 82272 81365 0.4
Mw 78650 78953 1.5
Mn 49383 50110 0.6
PDI 1.59 1.58 1.1
poly(methyl methacrylate co ethyl acrylate)
in THF
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ACQUITY APC Column Options
Particles based on the BEH chemistry
Five pore sizes – 45 Å (200 – 5,000) 1.7µm
– 120 Å (1,000 – 30,000) 2.5µm
– 200 Å (3,000 – 70,000) 2.5µm
– 450 Å (20,000 – 400,000) 2.5µm
– 900 Å* (TBD, later in the year)
Two surface chemistries – Organic solvent- XT
– Aqueous buffers – AQ
Three column lengths – 30 mm
– 75 mm
– 150 mm
Internal diameter: 4.6 mm
©2013 Waters Corporation – Seminari UPLC-MS 36
Traditional Gel-Based Stationary Phases versus Rigid Hybrid Particles
Gel-Based Stationary
Phases Rigid, Hybrid Particles
Must consider solvent compatibility
and have few solvent choices
Compatible with virtually any
solvent
Challenging to switch solvents Change solvents in one step
Columns must not dry out Columns ship dry
Collapse at higher flow rates and
sensitive to pressure &
temperature changes
Particles resistant to high flow
rate and high pressure
Wide selection of commercially
available columns (all MW ranges)
Column technology for low to
mid MW polymers
©2013 Waters Corporation – Seminari UPLC-MS 37
Introducing the ACQUITY Advanced Polymer Chromatography (APC) System
APC Isocratic Solvent Manager (pISM)
– Precise & reproducible flow rate
– Compatibility with challenging solvents
ACQUITY APC Sample Manager (pFTM)
— Less dispersion volume (tubing, valve)
— Solvent waste & vapour
management
— Compatibility with challenging solvents
Refractive Index Detector
— 1.3µl flow cell
— Stable & low dispersion
PDA, TUV & ELSD possible
Single Heating Zone CM (CM-S)
— Accommodate up to three 15cm col.
— Compatibility with challenging solvents
©2013 Waters Corporation – Seminari UPLC-MS 38
Empower Software with the
GPC option supports both
conventional data reduction (RI
only) and advanced data
analysis (MALS and
Viscometry)
This software remains one of
the strongest in the market
place and is often cited as to
why a Waters GPC solution was
purchased by the user
Empower GPC Option