part iii – polar compound retention supercritical fluid ... · part iii – polar compound...

©2015 Waters Corporation 1

Part III – Polar Compound Retention

Supercritical Fluid Chromatography (SFC)

Waters Meet-The-Experts Webinar December 2, 2016

Isabelle François, PhD, UPC²/SFC & Strategic Separations Business Development Europe & India


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

Polarity limits of chromatographic techniques

©2015 Waters Corporation 3 3



WebinarPart 1




WebinarPart 2




WebinarToday!


Fundamentals on supercritical fluid chromatography (SFC)

Practical aspects

– How does an SFC instrument work? – Parameters which influence separation

Application examples with focus on:

– Retention for polar compounds – Orthogonality in comparison to reversed phase liquid chromatography (RPLC)


Separation Technology Overview

Gas Chromatography

Liquid Chromatography

Supercritical Fluid Chromatography

Separation achieved by a temperature gradient

•High efficiency [N] • Virtually no limitation to column length

•Limited selectivity [α]

• Limited stationary phase options

Separation achieved by a solvent gradient

•Limited efficiency [N] • Limited due to pressure drop across column

• High selectivity [α] • Different modes: reversed-phase, normal-phase, SEC, IEX, affinity, ion pair, HILIC, GPC…etc.

Separation achieved by density/solvent gradient

•High efficiency [N] • Low viscosity allows longer columns and/or smaller particles

•High selectivity [α]

• Wide variety of stationary phase and mobile phase (modifier / additive) • Pressure and temperature as additional parameters to alter selectivity

GC

LC

SFC


History of SFC

• Introduced by Klesper et al. in J. Org. Chem. ‘High Pressure Gas Chromatography Above Critical

Temperatures’ • Initially performed on capillary columns

• First capillary SFC available on the market (Hewlett-Packard) based on work of Berger and Gere • CO2 only – density gradients • GC-like with applications limited to non polar compounds

• SFC is involved in the ‘green chemistry’ global effort • Acceleration of instrumental developments • 2012 – Waters introduces UPC²

• Extended to packed column SFC with improved and optimized instrumentation

• Introduction of polar modifier to CO2 • LC-like with development of applications for more polar

compounds


Advantages of a Supercritical Fluid in Chromatography

Data courtesy of Davy Guillarme, Jean-Luc Veuthey LCAP, University of Geneva, Switzerland


Why CO2 ?

Chromatographic technique similar to HPLC – Was introduced to replace NP -> unpolar solvent had to be mimicked by

supercritical fluid -> CO2 has very low dipole

Mobile phase is supercritical fluid + one or more co-solvents – CO2 is miscible with all organic solvents – CO2 is the most common supercritical fluid – MeOH is the most common co-solvent

Substance Critical Temp

oC Critical Pressure (bar)

Comments

Carbon Dioxide 31 74 Physical state easily changed

Water 374 221 Extreme conditions needed

Methanol 240 80 Extreme temperature needed

Ammonia 132 111 Highly corrosive

Freon 96 49 Environmentally unfriendly

Nitrous Oxide 37 73 Oxidizing agent


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

CO2 is compatible with LC detectors

CO2 is as a Green Solvent

– Environmentally neutral: • Recovered e.g. 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 as CO2 is a gas at room temperature

Interesting for prep scale!


CO2 Phase Diagram Critical point of CO2 Tc = 31.1°C Pc = 73.8 bar


CO2 Phase Diagram Critical point of CO2 Tc = 31.1°C Pc = 73.8 bar

But ... Almost all modern SFC is not supercritical!


Convergence Chromatography

Supercritical Fluid Chromatography

UPC²

SFC

Super Fluid Chromatography

Enhanced Fluidity Chromatography

Science Fiction Chromatography

Separations Facilitated by Carbon dioxide Caroline West @ HPLC2015 SFC short course

Unified Chromatography

SFC - What’s in a name?


What is SFC? Supercritical Fluid Chromatography? Convergence Chromatography?

SFC is like HPLC except the primary mobile phase is supercritical CO2 instead of an aqueous solution – CO2 is much less polar than water (similar to heptane) – Elution order is reversed (non-polar compounds elute first) – Organic solvent is used as a strong solvent (e.g. methanol, ethanol) - Modifier – Typical conditions are:

o Gradients go to typically 40-50% of modifier o Setting of back pressure is required o Flow rates are typically high in comparison to (UP)LC

• 1.5 – 2 mL/min for 3 mm id x 1.7 µm dp columns • Up to 4 mL/min for 4.6 mm id x 5 µm dp columns


• Column: Polar - SiO2, diol … • Mobile phase: (A) Heptane; (B) IPA Mostly isocratic • No MS compatibility

NPLC SFC RPLC

SFC Comparison with LC

• Column: Apolar – C18 • Mobile phase: (A) H2O; (B) MeOH Mostly gradient • MS compatibility

• Column: Polar - SiO2, diol, EP … Apolar – Cyano, C18 ... • Mobile phase: (A) CO2 ; (B) MeOH Mostly gradient • MS compatibility

• P and T as finetuning parameters – density control


SFC Comparison with LC

SFC

Like NPLC

Can provide identical separations but with ... – Better reproducibility – Fully MS compatible – Much faster — no long equilibration times – Minimal use of toxic organic solvents – Amenable to gradients (not only isocratic)

Orthogonal to RPLC

Typical RP applications can also be done but ... – SFC provides different/orthogonal separation – Similar reproducibility and robustness as UPLC – Fast analyses and equilibration – Ideal for scale up


Similar to UPLC – from hardware as well as operational standpoint, both fully MS compatible

ACQUITY UPC² ACQUITY UPLC Xevo TQD

SFC Instrumentation


ACQUITY UPC² ACQUITY UPLC Xevo TQD

Similar to UPLC – from hardware as well as operational standpoint, both fully MS compatible

SFC Instrumentation


Inject valve

Auxiliary Inject valve

Column Manager

PDA detector

Back Pressure Regulator (Dynamic and Static)

Waste Modifier CO2 Supply CO2

Pump Modifier

Pump

mixer Thermo-electric heat exchanger

How does it work? What is different? UPC² - Instrumentation


Inject valve


Column Manager

PDA detector



Pump Modifier

Pump




Inject valve


Column Manager

PDA detector



Pump Modifier

Pump


Make-up Pump

Mass Spec

Splitter



Flow rate

Additives

Diluent

Co-solvent/ modifier

Additives

Injection solvent Temperature

Back pressure

Flow rate

SFC Column

UPC² method development


Flow rate

Diluent

SFC-MS

Make-up solvent

Additives Flow rate

Interface

UPC²-MS method development


Stationary phases

2-ethylpyridine

Triazole

Amide

Silica

Propanediol

Aminopropyl

Cyanopropyl

PGC

Pentafluorophenyl

Phenylpropyl

Phenylhexyl

C4

C8

C18

RP-HPLC

SFC

Hig

h Lo

w

Polarity in SFC

NP-HPLC & HILIC

Solvents

Alkanes

Toluene

Dichloromethane

Chloroform

Acetone

Dioxane

THF

Ethyl acetate

DMSO

Acetonitrile

Isopropanol

Ethanol

Methanol

Water

Solv

ent

Stro

ng

Solvent strength with Supercritical

CO2

NP-HPLC

RP-HPLC & HILIC

UPC² method development


Binary Solvent Manager: – Mobile phase: o A: Supercritical CO2 o B: Methanol (MeOH)

– Flow rate: 1.8 mL/min – End time: 7 min – Gradient: see table

Column: – ACQUITY UPC² BEH 100 mm L x 3 mm i.d. x 1.7 µm dp – T = 40°C

Sample manager: – Strong wash: IPA – Weak wash: MeOH – Loop volume: 10 µL – Injection volume: 1 µL

Convergence manager: – P (outlet, APBR): 130 bar PDA: – Acquisition rate: 10 or 20 Hz

UPC² method development Typical method


– Column Selection o Stationary phase (interaction type)

• Achiral • Chiral

– Mobile Phase o Composition and type modifier o Isocratic/Gradient Mode o Additive o Flow rate

– Solute Properties – Pressure/Temperature – Density

A. Tarafder and I. François – ISC2016 – SFC Short Course

UPC² method development Parameters


Column has most influence on the separation SFC holds high orthogonality within the technology itself

: CSH PFP

AU

0.000

0.012

0.024

0.036

0.048

: HSS C18 SB

AU

0.000

0.012

0.024

0.036

0.048

: BEH HILIC

AU

0.000

0.012

0.024

0.036

0.048

: 2-EP

AU

0.000

0.012

0.024

0.036

0.048

Minutes0.00 0.60 1.20 1.80 2.40 3.00 3.60 4.20 4.80 5.40 6.00

ACQUITY UPC2 BEH 2-EP 1.7 µm

ACQUITY UPC2 BEH 1.7 µm

A B C(1,2)

D

G H

9

F

A B C

D

G H

9

F

A B

C D

G H

9 F

A B C

D G

H*

9

F

Unknown [M+H] = 266

Unknown [M+H] = 272

*Imp “H” is a broad peak under the unknown

Unknown [M+H] = 226

Unknown [M+H] = 226

ACQUITY UPC2 CSH Fluoro-Phenyl 1.7 µm

ACQUITY UPC2 HSS C18 SB 1.7 µm

UPC² method development Column

Identical methods and modifier, just changing the column!


C. West et al., The Column 10 (2014) 2-8

Column #17

Column #26

Column #40

Different selectivity

1

2 3 4

5

0 1 2 3 4

analysis time (min)



S. Khater et al. J. Chromatogr.A 1319 (2013) 148-159

CO2-MeOH 90:10 (v/v) 25°C 150 bar 3 mL/min

BEH silica HSS C18 SB

CSH Fluoro-Phenyl

BEH Shield RP18

BEH 2-EP

e

s

a -b

v



Torus DIOL • high density diol Torus 2-PIC • 2-picolylamine selector Torus DEA • diethylamine selector Torus 1-AA • aminoanthracene selector

O SiO

OO

OHOH

O SiO

OO

OHN

O SiO

OO

OHNH

O SiO

OO

OHNH

N

US 6,686035 US 7,223,473 Others patent pending

UPC² method development Column – Torus Column Technology


BZD 1. Diazepam 2. Midazolam 3. Flunitrazepam 4. Lormetazepam 5. Flurazepam 6. Alprazolam 7. Triazolam 8. Clorazepate 9. Bromazepam 10. Nitrazepam 11. Clonazepam 12. Oxazepam 13. Lorazepam 14. Clozapine 15. Olanzapine

N

N R2

R3 R4

R5

R1

150 x 4.6mm, 5µm 30bar

2 1 3

4

5 + 6 7

8 9

10 + 11

12 13 + 14

15 Pc = 63

450 x 4.6mm, 5µm

2 1 3

4

6 7 8 9 11 12

13 14

80bar

5 10 15

Pc = 108

Analytical conditions : CO2-MeOH gradient mode, 4mL/min ; PrincetonSFC 2EP 150 x 4.6mm, 5µm; Oven temp @ 40 C ; BPR @ 150bar ; UV @ 220nm


Reduced mobile phase viscosity and low pressure drops allow easy serial coupling and efficiency increase

UPC² method development Column length


Data courtesy of Davy Guillarme, Alexandre Grand-Guillaume Perrenoud, Jean-Luc Veuthey LCAP University of Geneva, Switzerland

Implementation of sub 2 µm particles aids in separation like in UHPLC Pressure increase is mild due to reduced viscosity of mobile phase From practitioner’s standpoint: go as fast as the instrument allows you! Van

Deemter is flat at elevated flow rates

UPC² method development Column particle size


C. West, Current Anal. Chem. 10,1 (2014) 99-120

0 20 40 60 80 100

Proportion of research articles where modifier is used (%)

MeOH

EtOH

iPrOH

nPrOH

ACN

Other

UPC² method development Mobile phase

Methanol is the most widely applied modifier in SFC today


Minutes 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

AU

0.00

0.20

0.40

AU

0.00

0.20

AU

0.00

0.10

0.20

0.30

AU

0.00

0.10

0.20

AU

0.00

0.10

0.20

AU

0.00

0.05

0.10

17% MeOH

15% MeOH

13% MeOH

11% MeOH

9% MeOH

7% MeOH

3.0 mL/min; 50°C; 1800 psi backpressure; 3.0x150 mm BEH 5 µm; ACQUITY UPC2

UPC² method development Mobile phase – isocratic mode


2 25% MeOH over 2.5min



3x100mm 1.7µm Torus Diol column

AUAU

AU

Time0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50

0.0

5.0e-2

1.0e-1

1.5e-1

0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.500.0

5.0e-2

1.0e-1

1.5e-1

0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.500.0

5.0e-2

1.0e-1

1.5e-1

Increasing co-solvent percentage in gradient methods Reduces elution and analysis time Provides peak sharpening and higher sensitivity In contrast to NPLC, gradients can be easily applied in SFC!!

UPC² method development Mobile phase – gradient mode


15

14 5 9

1312 3 6

1110

164 2

8

7

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00

15

14

5 913

12

63 11

410

16

2

8

7

AU

0.00

0.10

0.20

0.30

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00

Modifier MeOH (0.1% HCOOH)

Modifier MeOH/ACN 1/1 (0.1% HCOOH)

Courtesy of David Clicq, UCB, Belgium

3x100 mmx1.7µm ACQUITY UPC² BEH

UPC² method development Mobile phase – solvent selection


Evaporates quickly from bottle

43

– As in UPLC, additives are added to enhance peak shape – Typical SFC additives and concentrations are:

Additive Concentration

Basic NH4OH 0.1%

Ammoniumacetate / formate 10mM

Triethylamine 0.1%

Acidic Formic, acidic 0.1%

TFA 0.5%

Neutral Water 2%

Not compatible with MS

Cannot be too high as phase demixing might occur

UPC² method development Mobile phase additives

©2015 Waters Corporation 44 Grand-Guillaume Perrenoud et al., J. Chromatogr. A, 1262 (2012) 205

BEH 2-EP

N N N

SiO

SiO

SiO

Without additives With 20 mM NH4OH

0 < pKa < 4

6 < pKa < 8

8 < pKa < 11

Be careful with column resistance to strong bases !

UPC² method development Mobile phase additives


AU

AU

Minutes 1.4 1.5 1.6 1.7 1.8 2.1 2.2 1.3

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Butylparaben dissolved in diluents listed at right

Injection volumes:

0.5 (black), 1.0 (red), 1.5 (blue), 2.0 (green), 2.5 (light blue), 3.0 (pink), and 4.0 µL (brown).

AU

0.0

0.1

0.2

0.3

0.4

0.5

0.6 A

30/70 heptane/THF

B

Isopropanol

C

Methanol

Notice, there is some peak distortion for injection volumes of 0.5 µL!

Conditions Column: BEH 2-EP 5 µm 2.1x150 mm Method: 97/3 CO2/MeOH; 2 mL/min; 40°C; 2000 psi ABPR; 254 nm (350-450 nm compensation)

2.0 1.9

– Injection in weak solvent ensures optimal injection conditions

– For SFC, best are CO2-like solvents

UPC² method development Injection solvent


– Injection conditions in SFC are important – BUT ... Practicality is too!

– Solubility – Ease in sample prep – Small volumes of ‘polar’ solvents can be injected (even traces of

water) – Initial conditions of chromatographic separation can be adjusted

to force focusing at the head of the analytical column

UPC² method development Injection solvent

L. Novakova et al., Anal. Chim. Acta 853 (2015) 647-659

Doping agents in urine samples


Courtesy of Eric Lesellier, University of Orléans, France

– Although pressure and temperature have significant impact on density, these parameters are typically only used for finetuning

– Both parameters have most impact in the low modifier regime and less at higher modifier percentages

– Typically, higher pressures and lower temperatures reduce analysis times and ensure peak sharpening

– Temperature has higher impact on density but pressure is quicker to change

UPC² method development Pressure / temperature


AU

0.110

0.120

0.130

0.140

0.150

0.160

0.170

0.180

Minutes9.40 9.60 9.80 10.00 10.20 10.40 10.60 10.80

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

110 Bar

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Minutes7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00

AU

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

Minutes6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50

Original: 130 Bar AU

0.090

0.100

0.110

0.120

0.130

0.140

0.150

0.160

0.170

0.180

0.190

Minutes9.00 9.20 9.40 9.60 9.80 10.00 10.20 10.40 10.60 10.80

150 Bar

AU

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Minutes6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50

AU

0.110

0.120

0.130

0.140

0.150

0.160

0.170

0.180

Minutes9.10 9.20 9.30 9.40 9.50 9.60 9.70 9.80 9.90 10.00 10.10 10.20 10.30 1


AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00


AU

0.00

0.10

0.20

0.30

0.40

0.50

AU

0.00

0.10

0.20

0.30

0.40

0.50

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

40°C

50°C


UPC² method development Temperature

©2015 Waters Corporation 53 T. Berger, B. Berger, SFC 2012, Brussels, Belgium

SFC Applications

Status before UPC² launch in 2012 ...



UPC² Applications Status today and beyond …


Normal phase

Compounds with no retention in RPLC Compounds degrading

in H2O Orthogonal in

comparison to C18

Future scale-up

Vitamins

Simplified sample prep

Chiral = no-brainer Structural Isomers

UPC² Applications

Alternative to HILIC

Easy MS coupling!


Compounds with no retention in RPLC

Compounds degrading in H2O

UPC² Applications


Might be challenging to find alternative, more retentive RP column

As aqueous mobile phases are used, compounds might degrade at injection

HILIC is great to achieve higher retention, but column regeneration times are typically quite long

UPC² delivers instantly a different separation and many columns to select

UPC² uses non-aqueous mobile phases. When MeOH causes hydrolysis too, ACN can be used as modifier

UPC² is characterized by extremely fast separations and column regeneration times

UPC² is a great orthogonal tool to have in your laboratory when addressing challenging RP separations!


Separation of isomers

UPLC – 1 peak

UPC² – 2 peaks

UPLC – low retention + tailing

UPC² - higher retention + sharp peaks


Courtesy of Arjen Gerssen, Rikilt, Netherlands

Structural Isomers

Problematic compounds

in UPLC

Easy MS coupling!


Adenosine

Uracil

Sulfapyridine

Log DpH3 of -0.7

Log DpH3 of 0.0 Log DpH3 of -2.1

SFC hybrid silica HILIC bare silica

at pH 6

Very good retention of hydrophilic compounds in SFC.

Complementary selectivity.

RPLC at pH 9

SFC hybrid silica

HILIC bare silica at pH 6

RPLC at pH 9

SFC hybrid silica

HILIC bare silica at pH 6

RPLC at pH 9

2.0 mL of ACN

HILIC SFC 1.2 mL of

MeOH



Slide courtesy of D. Guillarme, University of Geneva J. Sep. Sci. 36 (2013) 3141-3151

©2015 Waters Corporation 59 Courtesy of Jason Hill, Waters Corporation

LSU

0.00

120.00

240.00

360.00

480.00

Minutes 0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60 4.00

1.7µm, 3 x 100mm 2PIC 1.4mL/min 35% MeOH 90C 3500psi ABPR ELSD detection


1. Fructose 2. Glucose 3. Sucrose 4.Lactose 5. Maltose

1

2

3

4 5

Food Sugar Mix


Courtesy of Michal Holcapek, University of Pardubice, Czech Republic

Workflow simplification

Lipidomics


Normal phase


• Separation of 30 polar and nonpolar lipid class standards within 6 min!

Nonpolar lipids

Polar lipids

• Conditions: column UPC² ACQUITY BEH (100x3 mm, 1.7 µm, Waters), positive-ion ESI-MS, 60°C, modifier MeOH/water (99:1) + 30 mM ammonium acetate, flow 1.9 mL/min, ABPR 1800 psi, standards of lipid classes

M. Lísa, M. Holčapek, Anal. Chem. 87 (2015) 7187

UHPSFC/ESI-MS of All Lipid Classes


Analyte

1. Vit A Acetate

2. Vit E Acetate

3. Vit K1

4. Vit A Palmitate

5. Vit E Tocopherol

6. Lycopene

7. Vit E Succinate

8. β-Carotene

9. Vit D3

10. Lutein Simultaneous analysis of fat-soluble vitamins and carotenoids in 10 minutes

Analyte LC mode Time

Vit A NP 12 min

Vit E NP 30 min

Lycopene NP 10 min

β-Carotene NP 10 min

Vit D3 NP 20 min

Vit K1 RP 12 min

Lutein RP 10 min

7 HPLC methods with total time ≈ 2 hours

SFC

HPLC


Easy MS coupling!

Replacing Multiple LC Methods


Vitamins in Food - Sample Workflow for HPLC

Infant Formula sample

Add D2 Int. Std. & Saponify

Hexane extraction

NPLC Analysis

RPLC Analysis Vitamin E

Vitamin A

Evaporate

Redissolve in MeOH

Evaporate

Redissolve in iPrOH

Semi-prep RPLC purification

Collect peaks

Evaporate

Redissolve in hexane NPLC Analysis

Vitamin D3

10 sample prep steps 3 analytical methods

3 instruments


Easy MS coupling!


3 sample prep steps 1 analytical method

1 instrument

Infant Formula sample

Add D2 Int. Std. & Saponify

Hexane extraction

Concentrate

SFC Analysis

SFC Analysis

Vitamin E

Vitamins A & D3

Vitamins in Food - Sample Workflow for HPLC


Easy MS coupling!


Orthogonal in comparison to C18


15

14 5 9

1312 3 6

1110

164 2

8

7

AU

0.00

0.20

0.40

0.60

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00


Pharma sample UCB on ACQUITY UPLC BEH C18 – 210 nm

Pharma sample UCB on ACQUITY UPC² BEH – 210 nm

Orthogonal in comparison to C18 Application at UCB – Comparison of current UPLC method

with UPC²


Courtesy of Alex Brien, Reach Separations, UK

Orthogonal in comparison to C18

Application at Reach Separations, UK Pharma CRO

UPLC

ACQUITY UPLC BEH C18

UPC²

ACQUITY TORUS 2-PIC

ADDITIONAL PEAK IN UPC²!


GC

LC

polarity

Log

MW

SFC

Which gap ? Application gap No Speed gap Yes Detection gap Yes ‘Mildness’ gap Yes Sustainability gap Yes

Hans-Gerd Janssen Unilever and professor @ University of Amsterdam, NL

Courtesy of Hans-Gerd Janssen, Unilever, Netherlands

UPC² Bridging the gap between LC and GC


Compound coverage in SFC!!

SFC does not have many unique applications. Chiral separation is (the only?) one. But SFC has many applications it can do ‘better’. SFC is faster, SFC is greener, Fine tuning is easier, etc. SFC can provide a frame of reference for GC: Do these labile compounds degrade? Are these adsorptive compounds lost? SFC has a very broad compound coverage.




SFC: broad distribution; LC: three series; GC: three series

Food emulsifier by NPLC, NP-SFC and GC

E3

E7

E7

SFC-MS

LC-MS

GC-MS

Compound coverage in SFC!

BPR gradient

1% modifier

BPR constant

Modifier gradient




Reduce Solvent Costs

Green Chemistry

Orthogo-nality

Increase Speed &

Throughput

Enantiomer isomer

Separations

Issues with Range of Polarity

Structural isomer

separations

Simplify Workflow

CONCLUSIONS

SFC / UPC²


Thank You!

You for your attention!

Acknowledgements

• Davy Guillarme, Alexandre Grand-Guillaume Perrenoud, Jean-Luc Veutthey, University of Geneva, Switzerland • Caroline West and Eric Lesellier, ICOA, France • David Clicq, UCB, Belgium • Hans-Gerd Janssen, Unilever, Netherlands • Alex Brien, Reach Separations, UK • Lucie Novakova, Charles University, Czech Republic • Michal Holcapek, University of Pardubice, Czech Republic • Arjen Gerssen, Rikilt, Netherlands

part iii – polar compound retention supercritical fluid ... · part iii – polar compound...

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