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


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


Which Supercritical Fluid ?

Courtesy of A. Grand-Guillaume Perrenoud, D. Guillarme, Pr J-L. Veuthey, University of Geneva


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.


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


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


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


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


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


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?


… in the ACQUITY Family


Gold Award at Pittcon !


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!!


Flow splitter and make-up pump are recommended when co-solvent % is less than 5%

Interfacing ACQUITY UPC2 with MS Detection



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


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)


Unified Classification

CO2/MeOH 90/10

Courtesy of C. West, E. Lesellier, ICOA – University of Orléans


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


Note Applicative


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



Questions ??


A Novel, Practical Approach to SEC/GPC Analysis

Introducing ACQUITY Advanced Polymer

ChromatographyTM (APC) System


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


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


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


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


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


New and Innovative Polymers

Polystyrene

Standard 510 Mp Alliance 2695/2414 6x150 HSPgel HR1

ACQUITY APC with RI 4.6x150mm; 45Å XT


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


Gel Based Columns vs Rigid Hybrid Particles

THF

DMF

Toluene

One System. One Bank of Columns. Solvent Flexibility.


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)


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


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


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


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


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

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UPC Sistema per la Convergenza Cromatografica · 2013-07-10 · Advancements in Gas Chromatography...

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