Exploiting New Agilent LC Technologies: Refined Solutions ... LC and SFC.pdf · Brand 2D-LC and SFC...

Brand 2D-LC and SFC Friday, September 14, 2012

Agilent Technologies, UHPLC User Summit 1

Exploiting New Agilent LC Technologies:

Refined Solutions 2D-LC and SFC

New flexible and user-friendly solutions for the most complex and difficult samples

Tony Brand, Ph.D.Senior Field ScientistSeptember 14th, 2012

1

The Van Deemter Plot: Advantages of Smaller Particles at High Linear Velocities

HETP

(cm

/pla

te)

-0.0005

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0035

0.0040

0.0045

5.0 m

3.5 m

1.8 m

2mL/min 5mL/min

Interstitial linear velocity (ue- cm/sec)0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

Unlike 5µm and 3.5µm columns, 1.8µm columns maintain higher efficiency at higher linear flows, which allows for significant gains in analysis speed. Typical Speed Gain: 5 – 10x



1998* 1.5 µm*(non-porous) 30,000

History of Commercial HPLC Particle Development

2003 1.8 µm 32,5002007/2008 2.7 µm (porous shell) 32,000**

Year(s) of Acceptance

Particle Size Most PopularNominal Size

Plates / 15cm(Approximate)

1950’s 100µm 200

1967 50 µm (porous shell) 1,000

1972 10 µm 6,000

GlassBead

12,0001992 3-3.5 µm 22,0001985 5 µm

2000 2.5 µm 25,000

* non-porous silica or resins ** 90-120 A pore

Irregularly-Shaped

Page 3January 2012

Liquid Chromatography

Mobile phase (flowing)

Mobile phase (flowing)

Column

MS

SolventsH20

ACN or MeOH

DegasserFlat membrane

PumpBinary

AutosamplerLoop Injector shown

UV Detector Mass Spec

Mix

ing

Stationary Phasecoated on ultra-high

purity silica

Steric Protection

Ultra-PureSilica

Selectivity Main Chain

Si OR

Si O Si

Si OR

PG R’

CH3CH

CH2 CH3

CH2 CH3CH

CH3A Stationary phase example: Zorbax Bonus-RP

Triple EndcappedSilanols for pH stability and good peak shape

1.7µm

0.5µm

0.5µm

Superficially PorousParticles



1995: The Agilent 1100: The LegacyStackable, comprehensive, flexible configurationsFront access for easy user maintenanceEffective leak managementErgonomic tubing organization for low delay volume and

bandspreadingSingle LAN/PC/HUB cable connectionFlexible handheld or PC/network control strategiesModular intelligence via the CAN networkEMF, Early Maintenance FeedbackColumn tracking via RF Tag

1290 Infinity

0

200

400

600

800

1000

1200

0 1 2 3 4 5

The Evolution of Power Range

bar

ml/min

Agilent 1100/1200, 400 bar 1995-2009 Standard LC

Agilent 1260, 600 bar 2009-2010 the New Standard

Page 6

Higher ResolutionHigher SpeedHigher Peak capacity/timeHigher SensitivityMethod TransferMore viscous solvents Lower Temperature



1290 Infinity Binary Pump- How it looks like inside

September 21, 2012Page 7

Channel A

ChannelBPurge

Valve

Solvent selectionvalve

Multi-layerHeat exchanger

Jet Weaver

Degasser

High ResolutionPump drives

1290 Infinity LC Binary Pump Technology- Maximum Power and Robustness

Page 8



F = 1.20ml/minT = 40°CAnalysis Time = 11minSolvent Cons. = 13.2ml

High Resolution:4.6mm x 150mm 5.0µm

min0 2 4 6 8 10 12

F = 4.80ml/minT = 40°CAnalysis Time = 1.05minSolvent Cons. = 5.1ml

High Speed:4.6mm x 50mm 5.0µm

min0 0.2 0.4 0.6 0.8 1

F = 1.00ml/minT = 40°CAnalysis Time = 1.1minSolvent Cons. = 1.1ml

High Speed & Resolution:2.1mm x 50mm 1.8µm

min0.2 0.4 0.6 0.8 10

The LC method and the Need for Speed….. Smaller particle columns, Higher Linear Flow Rates, Higher temperatures, Less Consumption => Lower Cost

Page 9

Max Speed at T = 95oC2.1mm x 50mm 1.8um

F = 2.40ml/minT = 95°CAnalysis Time: 0.4minSolvent Cons. = 1.0ml

PWHH = 197msec

min0.2 0.4 0.6 0.8 10

> 20x faster !

95%

5%

0 min, 5% Acetonitrile, 95% H2O… gradient ..12 min, 95% Acetonitrile, 5% H2O

January 2012

Agilent LC Valves: A New Era

1290 Infinity TCC(G1316C)

1290 Infinity Flexible Cube(G4227A)

1290 Infinity Valve Drive(G1170A)

2pos/ 6port2pos/10port4pos/10port6pos/14port8pos/ 9port

12pos/13port

StandardBio-inert

Micro-flow

600 bar1200 bar

Columns switching Solvent selection Fractionation Matrix-stripping Detector selection And........

Agilent QuickChange Valve Heads

Universal Valve Drive



Agilent QuickChange Valve heads

Kit PN Valve PN Description(as on valve label) Pressure [bar] Ports Positions

G4230A 5067-4107 8ps/9prt 600 bar 600 9 8

G4230B 5067-4121 8ps/9prt 1200 bar 1200 9 8

G4231A 5067-4137 2ps/6prt 600 bar 600 6 2

G4231B 5067-4117 2ps/6prt 1200 bar 1200 6 2

G4232A 5067-4144 2ps/10prt 600 bar 600 10 2

G4232B 5067-4118 2ps/10prt 1200 bar 1200 10 2

G4234A 5067-4146 6ps/14prt 600 bar 600 14 6

G4234B 5067-4142 6ps/14prt 1200 bar 1200 14 6

G4235A 5067-4147 12ps/13prt 200 bar 210 13 12

G5631A 5067-4148 2ps/6prt 600 bar 600 6 2

G5639A 5067-4134 4ps/10prt 600bar 600 10 4

n.a. 5067-4151 6ps/7prt 1200 bar CCV 1200 7 6

2ps-6prtvalve

2pos-10portvalve

6pos-14portvalve

8ps-9prtvalve

6ps-7prtvalve

In gray: not yet orderable

8-Position / 9-port valve headApplication example: Automated column selection

Page 12

• 2x 1290 Infinity Thermostatted column compartment

• 2x 8-pos/9-port valve head

• Up to 8 columns• Bypass or waste

positions can be configured

• For automated column selection



6-Position / 14-port valve headApplication example: Automated column selection

April 201013



• Up to 6 columns (4 columns recommended because of available space)

• For automated column selection

2-Position / 6-port valve headApplication example: Online sample enrichment

April 201014



• One enrichment and one analytical column

• For online sample enrichment

• Two pumps recommended

• Other applications like sample stripping, etc.



2-Position / 10-port valve headApplication example: Alternating column regeneration (ACR)

April 201015



• Two identical, analytical columns

• For alternating column regeneration

• Two pumps required!

1290 Infinity Therm. Column CompartmentNext level in usability:

New prolonged HX-carrierwith 3rd hand (not shown)

New valve drive and valve head

New capillary guide

New leak funnel

modified lowdisp HX

New door-open sensor

RFID-tag, stores type, pressure range, valve switches

NewThermalInsulation

New 2PS/6PT, 2PT/10PT and 9PT/8PS Quick-Change high pressure valves



2D-LC operation modes

Heart-cutting LC-LC

Time- or Peak-triggered

levelKnown samples/improving confidence: pharma, meth.-dev..

2D-LC: Injecting the efluent or a part of the effluent of one column to a second column, ideally with orthogonal separation behavior.Purpose: increase total separation power.

Comprehensive LCxLCStandard

orTime-/Peak-triggered

level

1D chromatogram

2D sampling

2D gradient

complex/unknown samples: bio-pharma, food, polymers....

18

Only peaks eluted from the 1st dimension column will be injected to the second column. Typically a peak from the first dimension will be sampled as a whole and a gradient with a longer run time than the collection time will be used. Also longer columns with higher seperation efficiency are being used in as 2nd dimension column.

2D-LC - Heart-cutting 2D-LC

LC1

LC2

Care must be taken if peaks are eluting from the first dimension column when a gradient on the second dimension is still running –this peak will be lost.

LC 1Pump

LC 2Pump

LC 1Autosampler

LC 2Detector

Waste

LC 1Detector



19

Only peaks eluted from the 1st dimension column will be injected to the second column. Typically a peak from the first dimension will be sampled as a whole and a gradient with a longer run time than the collection time will be used. Also longer columns with higher seperation efficiency are being used in as 2nd dimension column.

2D-LC - Heart-cutting 2D-LC

LC1

LC2

If peaks are eluting from the first dimension column when gradient on the second dimension is still running – this peak will be lost.

LC 1Pump

LC 2Pump

LC 1Autosampler

LC 2Detector

Waste

LC 1Detector

2D-LC - Comprehensive 2D-LC

20

The complete effluent of the 1st dimension column is injected onto the 2nd dimension column with very fast gradients. A peak of the first dimension should be sampled at least 3 to 4 times. The run time of the 2nd dimension method matches the collection time of the 1st dimension effluent. Finally, the peaks will be re-constructed.

LC1

LC2

LC1

LC2

1st peak from 1st dimension

2nd peak from 1st dimension

3rd peak from 1st dimension

LC 1Pump

LC 2Pump

LC 1Autosampler

LC 2Detector

Waste



2D-LC - Comprehensive 2D-LC

21

The complete effluent of the 1st dimension column is injected onto the 2nd dimension column with very fast gradients. A peak of the first dimension should be sampled at least 3 to 4 times. The run time of the 2nd dimension method matches the collection time of the 1st dimension effluent. Finally, the peaks will be re-constructed.

LC1

LC2

1st peak from 1st dimension

2nd peak from 1st dimension

3rd peak from 1st dimension

LC 1Pump

LC 2Pump

LC 1Autosampler

LC 2Detector

Waste

LC1

LC2

Example 2D-LC Application Scenarios

22

1st Mode 2nd Mode Advantage Disadvantage Column Examples

PROTEOMICS

Affinity Reverse Phase Orthogonality Low Peak Capacity, 1. AGILENT MULTIPLE AFFINITY REMOVAL

Off-line

Size Exclusion IonExchange Orthogonality Low Peak Capacity 1. PL-GEL (VARIOUS BEDS)

2. ZORBAX STRONG & WEAK ION EXCHANGE

Ion Exchange Reverse Phase Orthogonality Low Peak Capacity 1. ZORBAX STRONG & WEAK ION EXCHANGE

2. ZORBAX STM, POROSHELL

POLYMERS

Size Exclusion Normal Phase Orthogonality Low Peak Capacity 1. PL-GEL (VARIOUS BEDS)

2. ZORBAX CYANO, AMINO, SIL

Size Exclusion Reverse Phase Orthogonality Low Peak Capacity 1. PL-GEL (VARIOUS BEDS)

2. ZORBAX STM, POROSHELL

Reverse Phase Reverse Phase Miscible solvents, Strongly depends on column ZORBAX STM, POROSHELL

Broadest applications, and choice of mobile phase

Fast dual gradients, Highest peak capacity

Normal Phase Reverse Phase Orthogonality Solvent compatibility, 1. ZORBAX CYANO, AMINO, SIL

Limited application 2. ZORBAX STM, POROSHELL

PHARMACEUTICALS/METABOLOMICS

Compiled by Prof. Peter Carr, University of Minnesota



Hardware – Module-flexibility

23

1. Dimension

2. Dimension

1290 Infinity Binary Pump

1290 Infinity Binary Pump

1290 Infinity Autosampler or 1260

HiP Autosampler

Optional 1260/1290

Infinity Detector

One or two 1290 Infinity

TCC

Optional 1260/1290

Infinity Detector

1260/1290 Infinity

detector

1260 Infinity Binary or Quaternary Pump

1260 Infinity Capillary Pump 1260 Infinity

Autosampler

For 1st dimension

chromatogram and peak-triggering

To monitor waste-line

Almost any Agilent pump or autosampler in the 1st dimension with many different valve cobinations and detectors!

A 1290 Infinity Binary Pump for the 2nd dimension is required.

Hardware – Valve-flexibility

24

1. Dimension

2. Dimension

waste

Loop1

Loop2

2pos/10portvalve

Most other valve configurations for comprehensive and heart-cutting 2D-LC are supported as well. Easy transfer of existing 2D-LC methods!



Hardware – Valve-flexibility

25

1. Dimension

2. Dimension

waste

Loop1 Loop2

2x 2pos/6port valves

Also dual valve-head configurations supported with automatic synchronization of valve-drives!

Hardware – Valves, uniqueness and flexibility

26

1. Dimension

2. Dimension

wasteLoop1

Loop2

2pos/4port-duo

New Agilent-only special 2D-LC-QuickChange valve. Single valve with fully symmetric flow-paths and symmetric fill/flush-out behavior. Only valve that allows countercurrent flush-out of both loops.



Advantages of the new Agilent 2D-LC QuickChange valve head

All flow paths are equal (no additional bridging loops)Symmetric contercurrent fill/analyze direction of loops (reducing band-spread)

All in one valve (no synchronization, costs)

Loop1

Loop2

waste

2D-pump 2D-pump

1D-column

1D-column1D-column

Fill directionAnalyze direction

Valve

switching

1. 2.

Application examples-Advantage of shifted gradient features

Current state-of-the-art 2D-LC – narrow spread of peaks

RPLC x RPLCEasy method set-up but only little orthogonality

Data Analysis using GC Image LCxLC Edition Software from GC Image LLC.



29

• standard repeating with start- and end-time

• constantly shifted %B2D

• constantly shifted %B2D and shifted Δ%B2D

• Any combination

%B2D

Start 2D sampling End 2D sampling

%B1D

Time

%B2D

%B1D

Time

%B2D

%B1D

Time

%B2D

%B1D

Time

Comprehensive 2D-LC – gradient modesspecial 2D-gradient modes to improve resolution

30

Time-segmenting in Comprehensive 2D-LCReducing cost!

1D Method run time

1D Gradient/

2D Gradients

Idle flow rate

2D Pump flow rate

2D Pump flow rateSolvent saving!!!

2D Time-segments set in software idle idle idleTime-triggered Peak-triggered

Valve togglingIncrease life time!

%B (2D)

%B (1D)

F (2D)

1 D M

etho

d po

st-r

un ti

me

1D Chromatogram



31

Most easiest 2D-LC System Configuration- “One screen for the entire system”

Define 1D / 2D pump

Define detector in the second dimension

Define peak detector (optional)

Select the valve(s) to be used for 2D-LC

injection

Select a possible valve / loop

configuration

Graphical representation of the selected valve / loop configuration:• Flow path 1D & 2D• Animated valve switching

32

Select the 2D-LC mode: comprehensive

/ heart-cutting

Define the gradient of the 2nd dimension

Define repetition of 2nd dimension

gradient (Modulation time)

Show rollout of gradient in the 2nd dim

over the runtime of the 1st dimension

Define time window(s) where the

selected 2DLC mode is active

Most easiest set-up of complex 2D-LC methods- 2D-LC specific parameters of the 2D-pump

Graphical editing of gradient shift Access to standard

method UI of the pump

Operation values, warnings

Close-up of 2D-gradient

Solvent & Flow-Settings

Solvent consumption calculation



33

Example: graphical editing of a gradient shift – in less than a minute!- replace editing of large timetables by a few mouse operations

1 Use context menu to enter the editing mode

3 Draw a straight line by dragging the mouse to a new %B value at a specified runtime of the 1st dimension

2 Timetable entries are marked with circles

4 When releasing the mouse, a new TT entry is made and the gradient rollout is automatically updated

6 Insert / Delete shift points (mouse cursor and context menu changes near to a shift line)

5 Repeat step 3 + 4 with other TT entries

Resolution optimized!

Application examples-Advantage of shifted gradient features

Use of shifted gradient feature

Imagine programming this gradient manually! With Agilent 2D-LC Acquisition software a matter of a minute!

Before



Some Performance data- Testmix of 20 compounds – 2D-Precision

0.00

0.50

1.00

1.50

2.002D-Retention Time Precision RSD (%)

0.00

1.00

2.00

3.00

2D-Peak Volume* Precision RSD (%)

1st Dimension: Eclipse Plus RRHD, C18, 150 x 2.1 mm,

1.8 µm. Flow rate: 0.1 mL/min. Gradient: 0 min 5% B – 30 min 95% B,

40min stop-time.

2nd Dimension: Eclipse Plus RRHD, Phenyl Hexyl, 50 x

3.0 mm, 1.8 µm Flow rate: 3 mL/min. Initial Gradient: 0 min - 5% B, 0.5 min -

15% B, 0.51 min - 5% B, 0.65 min - 5% B. Modulation-time 0.65 min

15 compounds <1% RSD, all compounds <2% RSD

8 compounds <1% RSD, 16 compounds <2% RSD

*) not a peak area is measured but a three dimensional peak volume (intensity x 1D-time x 2D-time)!

Applications examplesPolyphenols from food matrix

36

1

2

34

56

7

8 9

10

11

1213 14

15

16

171819

202122

23

24

25 26

Compound RT 1st dim (min)

RT 2nd dim (sec) Peak Volume

Esculin mean 9.75 18.58 177,383s.d. nd 0.11 4,713RSD (%) nd 0.57 2.7

Rutin mean 13.65 33.68 72,375s.d. nd 0.07 853RSD (%) nd 0.22 1.2

Coumaric acid mean 13.00 25.57 660,541s.d. nd 0.13 13,037RSD (%) nd 0.52 2.1

Reservatrol mean 18.85 26.65 1,122,219s.d. nd 0.10 16,089RSD (%) nd 0.37 1.4

Salicylic acid mean 19.50 18.53 211,092s.d. nd 0.10 6,895RSD (%) nd 0.51 3.3

Luteolin mean 19.50 32.70 695,601s.d. nd 0.11 17,592RSD (%) nd 0.33 2.5

7-Hydroxy-Flavone mean 22.75 34.26 1,388,226

s.d. nd 0.10 17,195RSD (%) nd 0.30 1.2

Pinoslyvin mean 24.70 18,85 1,588,654s.d. nd 0.23 57,580RSD (%) nd 1.24 3.6

Chrysin mean 27.30 27.42 808,916s.d. nd 0.11 14,768RSD (%) nd 0.39 1.8

Flavone mean 28.60 26.35 1,008,012s.d. nd 0.13 20,911RSD (%) nd 0.48 2.1

Peak volume:all compounds <3.6% RSD



Application Examples-normal and stressed MAb, detection by ToF-MS

Intensity, cps

Gradient across analytical column 3-65% B

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340Time, min

0.00

2.00e6

4.00e6

6.00e6

8.00e6

1.00e7

Linear Salt Gradient 0-50% B

Step Salt Gradient to 100% B

Isocratic Salt Gradient at 100% B

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340Time, min0.00

2.00e6

4.00e6

6.00e6

8.00e6

1.00e7

Intensity, cps

37

Summary

38

The Agilent 1290 Infinity 2D-LC solution brings the power of two-dimensional LC-separation to you at a never before experienced ease-of-use!

Highest separation power by 2D-LC combined with outstanding performance of the 1290 Infinity LC – the ideal tool for complex samples form biological origin, polymers, food-extracs, and many more.

Innovative new hardware and software features for ease-of-use, reduced operation costs and highest performance.

Upgradability or re-use of existing Agilent LC equipment.



Supercritical Fluid Chromatography:A New Era

Tony Brand, Ph.D.Senior Field ScientistAgilent Technologies, Inc.

Page 39

The Supercritical State of a Solvent

• Above a critical pressure liquids cannot enter into the gaseous state

• Above a critical temperature gases can no more enter into liquid state

• Above both, the critical temperature and the critical pressure solvents are in a supercritical state

Gas- and Liquid like properties

Page 40



Created by Christy Aurigemma, Pfizer, La Jolla

Page 41

Agilent 1260 600bar Binary pumpLeft side of Agilent binary pump exclusively meters CO2Right side: Selectable Modifiers

The Agilent 1260 SFC SystemLeveraging LC Technology

One extra setting:

BPR pressure

Agilent ALS or Hi-PALS autosamplersModified to use a loop injection

Agilent 1260 Thermostated Column CompartmentsUp to 8 columns, up to 250mm columns with multiple modules

Low

cos

t bev

erag

e gr

ade

CO

2

Agilent 1260 Diode Array DetectorHigh pressure flow cell

Put

ting

a st

anda

rd L

C

unde

r pre

ssur

e

Page 42



Phase diagram of pure CO2

with densities. Dashed lines represent approximate constant density lines

0

50

100

150

200

250

300

350

400

-70 -20 30 80

Temperature, °C

Pres

sure

, bar

0.041

0.86

0.62 0.29

0.098

1.120.99

0.83

0.720.941.09

1.06

0.91

0.84

0.96 0.891.02

0.59

0.19

0.75

0.82

0.77

0.89

0.95

0.991.14

0.0880.113

0.97

1.11

1.09

1.06 1.02

1.02

1.02

1.001.05

0.920.94

0.98

1.05

0.79

0.78

0.87

1.08

0.28

0.29

0.105

0.191

CP

Constant Density Lines: No abrupt changes

43

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 100 200 300 400

Ref

ract

ive

Inde

x

Pressure, Bar

CO2 at 40°C

Water at 20°C

The Refractive Index of CO2

varies Much More than Water

1

1.05

1.1

1.15

1.2

1.25

0 100 200 300 400 500

Ref

ract

ive

Inde

x

Pressure, Bar

40°50°60°70°

varies Significantly with Temperature

Page 44



Agilent 1260 Analytical SFC Solutions Unparalleled performance

< 0.02 amu noiseUltra-fast, ultra-simple

Lowest running cost of any LC or SFC!uses food grade CO2

minimal waste disposal costs

Standard Agilent softwareChemStationMassHunter

Agilent service and supportValidation services, IQ/OQ, etc

Page 45

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 20 40 60 80 100

Diff

Co.

, cm

2/se

c x1

05

% Co-solvent

60oC50o

40o

CO2/MeOH

C. Mantell, M. Rodrı´guez, E. Martı´nez de la Ossa. “Measurement of the diffusion coefficient of a model food dye (malvidin 3,5-iglucoside) in a high pressure CO2+methanol system by the chromatographic peak-broadening technique”, J. Supercrit. Fluids 25 (2003) 57–68

SFC is Faster Because Diffusion Coefficients are Larger

SFC Region

HPLC Region

VanDeemter eq.

HETP = A + BD1,2/µ+ Cµ/dp2D1,2

46



0.5

0.7

0.9

1.1

1.3

1.5

1.7

1.9

2.1

2.3

2.5

0 0.2 0.4 0.6 0.8

Linear Velocity, cm/sec

Red

uced

Pla

te H

eigh

t

Kinetex 2.6µm 150 x 4.6mm; methanol in CO2, 150 bar, 50°C, 0.3(?) µL injected.

30% MethanolH = A + BD1,2/µ+ Cµ/dp2D1,2

hr =H/dp

≈ 33,000 plates

SFC Can Deliver:Extreme Efficiency at High Flow Rates

47

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Mole fraction organic

, m

Pa-

s

CO2

H2O1.0

0.5

1.5

0

2.0

2.5

3.0

MeOH

EtOH

aqueous data from: J. Chem. Thermodynamics, 39 (2007)1578-1588

ΔP = c LµeT/dp2

Pressure Drop-Viscosity

Carbon dioxide has less than 1/10th the viscosity of water.When water is mixed with alcohols the viscosity actually increases.

48



0

50

100

150

200

250

300

350

0 20 40 60 80 100

% Modifier

Pres

sure

inlet pressure

outlet pressurePure CO2ΔP= 9 bar

Pure MeOHΔP= 250Bar

The pressure drop across columns in SFC is much lower than the pressure drop in reversed phase HPLC due to much lower viscosities.

Pressure Drop-Viscosity

4.6x250mm, 5µm, 2ml/min, 40oC

49

0

50

100

150

200

250

300

350

400

450

0 0.5 1 1.5 2 2.5 3 3.5

Flow Rate, ml/min

Col

umn

Hea

d Pr

essu

re, B

ar

ΔP

3x100mm, 1.8µm Zorbaz RX-Sil column, 1.5ml/min of 22.5% methanol in CO2;

150Bar outlet pressure, 50oC. As the flow rate goes up the tubing becomes more restrictive.

Even at High Flows, the DP can be Modest

50



0

50

100

150

200

250

300

350

400

450

0 10 20 30 40 50 60 70

% Methanol

Col

umn

Head

Pre

ssur

e, B

ar

bottom: 1.5ml/min; top 2ml/min; 3.0x100mm RX-Sil 1.8µm column @ 50oC, 150bar outlet pressure

Column Inlet Pressure:A 3x100mm, 1.8µm Column

51

Higher speed/throughput - more samples/day; more rapid re-equilibration - shorter cycle time compared to reversed phase HPLC

Low pressure drops - High resolution - doesn't require ultra-high pressure pumps, etc. due to low viscosity and high diffusion

Dramatically lower operating cost. Low solvent consumption/low waste generation. Green!

Orthogonal to reversed phase HPLC, comparable to normal phase

Great for isomers, including enantiomers

High Sensitivity

The Aurora A5 modules allows conversion of an HPLC into an SFC!

Practical Advantages of SFC?

52



SFC issues ….

Carbon Dioxide is TOXIC at levels above 15%. A CO2

detector/alarm is <$200 on the internet!

In Reverse Phase LC, a 50mm x 3.0mm sub-2 micron or Poroshell C18 is the standard. For more polar compounds, try C8, then C4 and then a Aqua column. In SFC, there is no similar “progression” of columns.

The true application space is still being mapped! Can on-line SFE be combined with SFC in a single instrument for complex matrices (e.g. plasma, urine, etc.)?

85% CO2 15% MeOH, 4.75ml/min, 150bar, 50°C, Kinetex 4.6x150mm, 2.6µm HILIC

0.4

-1

0

1

mAU

0.8 1.20

min

SFC is Sensitive

N ≈ 25,000 plates

Noise ≈ 0.07mAU

W1/2 ≈ 0.003-0.0045min (80Hz) filter

54



14 sec

10 chromatograms/ 6minutesDP < 230 bar

SFC is Super Fast Chromatography

4.6x50mm, 1.8µm Zorbax RX-Sil 30% methanol at 5ml/min, 150 bar out, 50oC ibuprofen, ketoprofen, caffeine, theophyline, theobromine

55

56

Analytical SFC applications



Can SFC Separate My Compounds?

Any solute soluble in methanol or a less polar organic solvent will elute in SFC (>80% of small drug molecules)

Strong organic acids and bases, require a modifier and an additive in the mobile phase

Most salts of organic acids and bases elute

Smallish peptides elute

Solutes with mass <25,000 can be separated with carbon dioxide based fluids

57

Solutes requiring an aqueous environment

Solutes requiring a buffered or ionic aqueous environment, i.e. biomolecules such as proteins will NOT elute (maybe)

Most Inorganic salts

Solutes with mass >25,000 mw will not elute with carbon dioxide

SFC-MS: PAH Mix 25 (100ppm)

Nacalai π-NAP (4.6x150 mm, 5 µm), Injection = 5 µL, Flow Rate = 2.0 mL/min @ 120bar, SF = CO2, Mod = MeOH with 2% H2O, Gradient = 0-20min: 5-30%, Col Temp = 40oC, Caloratherm = 60oC,Make-up Flow = MeOH at 0.2 mL/min, MS = APCI, Cap V = +3000V, Corona I = 4.0µA, Fragmentor = 200V, Drying Gas = 5.0 mL/min @ 325oC, Nebulizer = 60 psig, Vaporizer = 450oC

UV 254 nm

MS scan, APCI, 80-350 amu

1 2

2

4

5,6

57

8

98

9

10

10

11

1112

12

13

14

14

1516

1615

13

7

3

3

Evaluation of SFC-MS Configurations for the Analysis of Lipids, Sterols, and PAHs,M. Dunkle, A. dos Santos Pereira, F. David, P. Sandra, SFC 2011 poster

1 Naphthalene (FW 128)2 Acenaphthylene (FW 152)3 Acenaphthene (FW 154)4 Fluorene (FW 166)5 Anthracene (FW 178)6 Phenanthrene (FW 178)7 Fluoranthene (FW 202)8 Pyrene (FW 202)9 Benzo(a)anthracene (FW 228)10 Chrysene (FW 228)11 Benzo(k)fluoranthene (FW 252)12 Benzo(b)fluoranthene (FW 252)13 Benzo (a)pyrene (FW 252)14 Dibenzo(a,h)anthracene (FW 278)15 Indeno(1,2,3-cd)pyrene (FW 276)16 Benzo(g,h,i,)perylene (FW 276)



SFC-MS: Lipid Mix 100ppm

Separation Conditions: Column Zorbax Eclipse XDB-C18 (4.6 x 150 mm, 5 µm), Injection = 5 µL, Flow Rate = 2.0 mL/min, Outlet P = 120 bar, SF = CO2, Mod = MeOH with 2% H2O , Mod gradient = 0-15min: 5-50%, Col Temp = 40°C, Caloratherm = 60oC, Make-up Flow = MeOH at 0.2 mL/min. MS = APCI scan (450-1000 amu), MS = APCI, Cap V = +3000V, Corona I = 4.0µA, Fragmentor = 70, Drying Gas = 10.0 mL/min at 325oC, Nebulizer = 60 psig, Vaporizer = 350oC

min2 4 6 8 10 12 14

0

100000

200000

300000

400000

500000

600000

MSD1 TIC, MS Fi le (C:\CHEM32\1\DATA\ORE11138_20110504_LIPIDS\ORE11138_PAH 2011-05-04 08-56-47\201105040000003.D) AP

PPP

OOO

POS

m /z5 00 6 00 70 0 80 0 9 00

0

2 0

4 0

6 0

8 0

10 0

* MSD 1 SPC, t ime= 1 0.74 4 of C:\C HEM3 2\1\DA TA\O RE 111 38 _2 011 05 04 _L IPIDS \O RE1 11 38 _PAH 20 11-0 5-04 08 -5 6-4 7\20 11 05 040 00 00 03.D

Max: 243328

903

.9

887

.8

605

.6

886

.8 8

85.

8

604

.6 6

03.6 OOO

[M-RCOO]+

[M+H]+

m /z55 0 600 650 7 00 7 50 8 00 8 50

0

2 0

4 0

6 0

8 0

10 0

* M S D 1 S P C, t im e= 8 .8 24 o f C : \ CH E M 32 \ 1\ DA T A \ O RE 1 113 8_ 20 110 50 4_ LI P I DS \ O RE 11 13 8_ P A H 2 01 1-05 -04 0 8-56 -47 \ 201 10 50 400 00 00 3. D

M ax : 30 2 0 0

824

.8

553

.6 5

52.6

551.

5 PPP[M-RCOO]+

m /z60 0 65 0 70 0 7 50 80 0 8 5 0 90 0

0

2 0

4 0

6 0

8 0

10 0

*MS D1 SP C, t im e= 11 .28 8 of C:\C HEM3 2\1 \D ATA\O RE1 11 38 _2 011 05 04 _L IP ID S\O RE1 11 38 _PAH 20 11- 05 -04 0 8-5 6-4 7\20 11 05 040 00 00 0 3.D

Max: 25608

58

1.6

890

.8 607

.6

862

.8

87

9.9

878.

9

58

0.6

578

.6

861

.8

606

.6

579

.6 5

77.6

605

.6 POS[M-R1COO]+

[M-R2COO]+

[M+H]+


Steroids by LC-APCI-MS and SFC-APCI-MS

1 Vitamin E2 Cholesterol3 Stigmasterol (3)4 β-sitosterol

Vitamin E spectrum by SFC/MS

Vitamin E spectrum by LC/MS

Zorbax Eclipse XDB-C18 (4.6x150mm, 5µm), Injection = 5µL, Flow Rate = 2.0 mL/min (SFC), 1.0 mL/min (LC), Outlet P=120 bar, A: CO2, B: MeOHw/ 2% H2O (Isocratic at 5%), MP= ACN/i-PrOH (5:4) isocratic, Temp = 40oC (SFC), 50°C (LC), Caloratherm = 60oC (SFC), Make-up = MeOH @ 0.2mL/min (SFC). MS = scan 350-500amu., Cap V = +3000V, Corona = 4.0µA, Drying Gas = 12 mL/min @325oC, Neb = 50 psig, Vap = 350oC.


UV sensitivity similar for LC and SFC

MS sensitivity better for SFC/MS

MS spectra “cleaner” for SFC/MS



SFC Separation of Nucleosides and Nucleic Acids

urac

il aden

ine

aden

osin

e

cyto

sine

guan

ine cytid

ine

guan

osin

e1 2 3 minutes

UV abs

Several not soluble in either MethanolOr water. Requires Acid or base to dissolve

4.6 x 200 mm Hypersil Amino

Methanol/water/ammonium acetate/

formic acid In carbon dioxide

50°C, 160 bar

Thia

min

e m

onon

itrat

e

Nia

cina

mid

e

Rib

ofla

vin

Nia

cin

Vita

min

B 1

2

1 2 3 4

SFC Separation of Water Soluble Vitamins



1 2 3 4

Vitamin B12

Methanol + 10% water + formic acid + ammonium acetate

SFC-MS spectraVitamin E

Separation Conditions: Column Zorbax Eclipse XDB-C18 (4.6 x 150 mm, 5 µm), Injection = 5 µL, Flow Rate = 2.0 mL/min (SFC), 1.0 mL/min (LC), Outlet P = 120 bar, SF = CO2, Mod = MeOH with 2% H2O (isocratic at 5%), MP = ACN/Isopropanol (5:4)isocratic, Col Temp = 40°C (SFC), 50°C (LC), Caloratherm = 60°C (SFC), Make-up Flow = MeOH at 0.2 mL/min (SFC). MS = APCI scan (350-500 amu), MS = APCI, Cap V = +3000V, Corona I = 4.0µA, Fragmentor = 70, Drying Gas = 12.0 mL/min at 325°C, Nebulizer = 60 psig, Vaporizer = 350°C

m/z320 340 360 380 400 420 440 460 480

0

20

40

60

80

100

*MSD1 SPC, time=4.325 of C:\CHEM32\1\DATA\ORE11138_20110502_PAHS\20110502000009.D APCI, Pos, Scan, Frag: 70

Max: 177024

427

.4

433

.4

429

.4 4

32.4

431

.4

[M+H]+SFC-APCI-MS1) Vitamin E



13mM ammonium trifluoroacetate in methanol 5% for 1min, then 5%/min to 50%, hold 5 min, 2ml/min, 120 bar outlet, 40oC,Ethylpyridine 4.6x250mm, 5µm (Princeton)

H3C

H3C

NO

N

O

O

O

N

O

H3CCH3

N

O

H3C

CH3

NO

CH3

CH3

N

O

N

N

N

NO

N

O

N

O

N

O

N

O

H3C

N

O

CH3

N

O

NO

N

O

NO

N

O

N

N

O

OO

N

O

N

N

O

OO

N

O

NO

N

O

N

N

O

OO

N

O

N

O

OO

N

O

N

O

S

CH3

N

O

N

N

O

N

NN

N

OH3CCH3

NO

CH3

CH3N

O

O

O

NO

CH3

CH3N

O

O

NO

CH3

CH3N

O

O

O

N

O

N

O

O

N

O

N

O

N

O

NO

N

O

O

N

H3C

CH3

H3CCH3

H3C

CH3

H3C

CH3

CH3

O

N

O

H

H

H

H

H

H

H

H

H H

H

H

H

H

H

HH

H

H

H

H

H

H

H

H

H

HH

H

H

H

HH

H

H

H

H

H

H

HH

H

H

H

H

H

H

H

H

HH

H

H

H

HH

H

H

H

H

H

HH

H

H

H

H

H

H

H

H

H

H

H

HH

40mer

J. Zheng. et al., Anal. Chem.,78, 1535-1545 (2006)

Peptides: A Limited Number of Reports

65

What Didn't We Cover That Is Important?

Surfactants– ethoxylates/proproxylates

UrethanesSilicone oilsFlavanoids/anti-oxidantsSweetenersMost pesticides/Herbicides

– carbamates– phenylureas– sulfonylureas



HPLCWaste

Hybrid System in SFC Mode

123

4

5

6 7

8

9

10

SFC-Binary Pump

BPR

Loop back Restrictor

SFC Pump outlet

LC Pump:G1310B orG1311B orG1312B LC

Pum

p outD

etector out

SFCWaste

68



Waste

123

4

56 7

8

9

10

BPR

Restrictor

SFC Pump inlet

LC Pump:G1310B orG1311B orG1312B LC

Pum

p outD

etector outSFC Pump outlet

SFC-Binary Pump

The Agilent Hybrid SFC/LC SystemUHPLC Mode

69

Advantages of the Agilent Hybrid SFC/UHPLC

Orthogonal method screening in one single system

Switch from SFC to UHPLC forth and back in a single sequence

No equilibration time between LC and SFC

Significant cost saving, only one system has to be purchased

Direct results comparison between SFC and LC

No instrument to instrument variation

Save lab space

The only system which can offer both techniques in single system

70



Performance: Overlay SFC versus UHPLC

71

SFC

LC

1 - Caffeine 2 - Theophylline3 - Cortisone 4 - Prednisone 5 - Hydrocortisone 6 - Prednisolone7 - Sulfomerazine8 - Sulfaquinoxaline

Repeatability: SFC and UHPLC in a single sequence

72

Part of a sequence with alternating SFC and LC batches

SFC-Mode LC-Mode



Application: PAH with the Hybrid SFC/UHPLC System

min0 2.5 5 7.5 10 12.5 15 17.5 20

mAU

0

10

20

30

40

50

DAD1 A, Sig=254,16 Ref=360,100

min1 2 3 4 5 6 7

mAU

0

20

40

60

80

DAD1 A, Sig=254,16 Ref=360,100

PAH- mixAcenaphteneAcenaphthyleneAnthraceneBenzoanthraceneBenzo(a)pyreneBenzo(b)flurantheneBenzoperylenBenzo(b)flurantheneBenzo(k)flurantheneChryseneDibenzoanthraceneFluorantheneFluoreneIndenopyreneNaphtalenePhenanthrenePyrene

LC-mode

SFC-mode

Column: Zorbax Eclipse PAH (4.6x 150 mm, 5 um)mobile phase A : H2O, B: ACN

Gradient:

time %B

0 40

20 95

21 95

21.5 40

Column: Zorbax Eclipse XDB C18 (4.6x 150 mm, 5 um)Mobile phase A: CO2, B: MetOH, 2% H2O

time %B

0 5

10 22.5

11 60

11.5 5

Gradient:

73

SFC uses Normal Phase SeparationOrthogonal to reverse phase HPLC

Trace contaminants elute in the order 1, 2, 3, 4, 5, 6 in SFC,but 6, 5, 3, 2, 1, 4 in HPLC

In general,

More polar componentselute last in SFCelute first in LC

Least polar componentselute first in SFClast in SFC

SFC

LC



SFC is Normal PhaseWith None of the Problems of Normal Phase HPLC!

No problems with water

• stable retention times• rapid re-equilibration• can add water to the modifier as an additive• can inject partially aqueous samplesGradient elution is typical

Provides nearly the opposite retention of reverse phase HPLC.

• enhanced purity assessment• improved trace analysisMultiple mobile phase parameters for changing retention and selectivity

Wide choice of columns to change selectivity and retention

75

Everything you need to know about SFC in 1 slideOr…. Top 10 things to remember… Think normal phase LC, Liquid CO2 is much like heptane. Well established for chiral separations. Usually performed as gradient elution, changing the composition of the mobile phase vs time

with a methanol/ethanol/isopropanol more polar co-solvent (0-60%, no benefit going higher!) Use polar stationary phases such as bare silica, cyano, amino, diol, ethylpyridine, “Premier”,

and others. Liquid CO2 is MUCH less viscous than organic solvents so delay volume does not cause

dispersion! Dispersion due to thermal changes is VERY significant! PEEK tubing is better than

stainless steel. Try to insulate tubing between devices and the column. If it is not hissing and not cold, icing, or condensing atmospheric moisture, it ain’t broke……

so there is another reason for not having a signal. Flow rate 1.5-5.0mL/min, go lower than 1.5mL at your own risk! The most critical setting is Back Pressure for the Back Pressure regulator. Default setting is

200bar, 160bar for SFC/MS since some pressure is bled off with the “leak” to the MS. MS requires APCI, Electrospray requires a make-up solvent containing a volatile buffer (e.g.

formic acid in methanol).



Practical Advantages of SFC

FAST CHROMATOGRAPHY for non-volatile compounds! higher speed/throughput--more samples/day more rapid re-equilibration--shorter cycle time compared to

reversed phase HPLC Orthogonal to reversed phase HPLC Great for stereo-isomers, chiral compounds High Sensitivity Low pressure drops-High resolution Low solvent consumption/low waste generation An alternative to normal phase LC Lower operating cost Green!

More Information on...

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• Application Notes: • Chiral impurity analysis(5990-5969EN)• Achiral analysis (5990-6413EN)• Sub 2um analysis for ultra fast separation (5990-6412EN)• Highest resolution separation (5990-6934EN)• Strategies for column and mobile phase selection

with the Agilent 1260 Infinity SFC System (5990-7147EN)• The Agilent SFC/MS solution (5990-7972EN)• Separation of enantiomers and conformers of Tofisopamon (5990-9315EN)• Enantiomer separation of nonsteroidal anti-infl ammatory drugs (5990-9459EN)• Agilent 1260 Infinity Hybrid SFC/UHPLC System (5990-9514)• Determination of polymer additives and migration products prevalent in food packaging

material (5990-9598EN)• Determination of phthalate migration from toys (5990-9597EN)

• SFC cost calculator: calculates operating costs of SFC vs. LC• Hybrid SFC/UHPLC Video• Hybrid SFC/UHPLC E-seminar• SFC Compliance Primer



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