The Past, the Present

and the Future(?)

of SFC

by:

Terry A. Berger, PhD, DIC

SFC Solutions, Inc.

in association with

Agilent Technologies

lets talk:

tabergersfc@aol.com

Why sfc?

Best technique for chiral.

Faster, higher resolution,

Normal phase but 3-5X faster rHPLC

Pressure drops 1/3rd to 1/5th HPLC.

400-600 bar systems fine

Very wide application space.

Ideal for hydrocarbons to primary aliphatic amines. Even small peptides.

Both aqueous and oil based samples can be injected with minimal sample prep.

Water not a problem-fast equilibration

Green technology-

the CO2is recycled

much lower volume of toxic solvent waste

lower disposal cost

Much cheaper than rHPLC solvents. Euro 0.1-1.0/Kg for CO2

CO2 Safety

fire extinguisher.

CO2is a product of human respiration-

soda machines in every cafeteria

Easy MS interface

Past, Present and Future(?)

• The Past was rather rocky and unclear- “Murky

Research” Stage

– It spanned from 1958 to at least (!)1992. (34 years!)

• The Present- Beginnings of Industrial Acceptance

– Started in 1992 with 2nd generation instruments

• The Future(?) – Mainstream use?

– UHPSFC has arrived

– SFC replaces 40-60% of HPLC?

1870’s

Some inorganic gases, surprisingly, acted as solvents above their

critical point

Hannay and Hogarth 1879

Jim Lovelock

• Proposed using dense inorganic gases as

SOLVENTS to separate heavy, non-volatile solutes

in 1958

• Designed the GC that analyzed the Martian atmosphere

as part of Viking

• Invented the ECD, and argon ionization detectors for GC

• Developed the Gaia hypothesis that the earth is a living

organism

Ernst Klesper did first experiments as a post-doc at Johns

Hopkins

• just a preliminary study, never finished

• separated metal porphorins

• chlorofluorocarbons at 230°C

• very large particles

• No pump. Heated the fluid in an enclosed space to generate a head pressure

• no detector!– collected fractions and analyzed off-line!

• No back pressure regulator. needle valve?

• Called it High Pressure Gas Chromatography– above the critical temperature

– (but below the critical pressure)

3 page Communication with the Editor. E. Klesper, A.H. Corwin, D.A.Turner, (1962) J. Org. Chem., 27, 700-706

Sie and Rijnders, Shell Amsterdam

immersed CO2 tank in water

bath and heated the water!

metering valve on outlet could

tolerate 5Kg/cm2

sometimes used CO2 or pentane

They don’t appear to know

about Klesper (obscure ref?)

They were below the critical

pressure at the outlet

S.T.E. Sie, GWA Rijnders, High Pressure Gas

Chromatography and chromatography with

Supercritical Fluids. 1. The Effect of Pressure

on Partition Coefficients in Gas-Liquid

Chromatography with Carbon Dioxide as the

carrier Gas”, Separation Science, 1 459-490 (1966)

Control Column Inlet Pressure

Very Large particles

control flow with metering valve

water bath!

metering valve

J. Calvin Giddings

“Ultra-High-Pressure Gas Chromatography

with Micro Columns to 2000 Atmospheres”

Separation Science 1, 761-776 (1966)

packed columns 500µm ID 2-3meters long

packed with 13µm particles

pump acted as a pressure source

amplifier pump

outlet pressure was NOT controlled

CO2 and NH3

Pre-eminent chromatographic theoretician of his time

J. Calvin Giddings

Proposed that very dense fixed gases

like CO2 were much stronger solvents

than previously thought

Published in Science:

J. Calvin Giddings, M.N. Myers, L.McLaren, R.A. Keller

“High Pressure Gas Chromatography of Nonvolatile

Species” Science 162, 67-73 (1968)

He was WRONG!!!!

This mistake had devastating consequences

for multiple DECADES

He did not know column outlet pressure

Didn’t include a BPR (pressure reducing valve) until 1970

Czubryt, Myers, Giddings, J. Phys. Chem., 74,

4260-4266 (1970)

MeOH

ACN

EtOH

IPA

CH2Cl

2

CCl4

Pentane

MeOH

EtOH

IPA

EtAc

CCl4

Pentane

CO2

ACN

P` δ + Giddings

CH2Cl

2

MeOH

EtOH

ACN, IPA

CCl4

CH2Cl

2

EtAc

40% MeOH

20% MeOH

10% MeOH

Pentane

CO2

ENRε°, silica

MeOH

EtOH

IPA

ACN

CH2Cl

2

Pentane

CCl4

Reality Check: Out of Context to later understanding

Giddings was wrong

Jentoft and Gouw, Chevron Research, Richmond CA

Pressure-Programmed Supercritical Fluid Chromatography

of Wide Molecular Weight Range Mixtures” (1970) J. Chromatogr.

Sci. p 138-142.

Pressurize pentane with N2

programmed pump pressure,

NOT outlet pressure

microregulating valve just

before detector

Jentoft and Goaw

The pump sets the column

head pressure

A “Micro-Regulating” valve

is used to control Flow Rate

What is the outlet pressure?

Milos Novotny

M. Novotny, W. Bertsch, A. Zlatkis

“Temperature and Pressure Effects in

Supercritical-Fluid Chromatography”,

J. Chromatogr. 61, 17-28 (1971)

varied particle size

measured inlet and outlet pressure

restrictor for flow control (no BPR)

Conclusion: pressure drops are bad for efficiency

small particles are worse than larger particles (!)

This conclusion was important and unfortunate

J.A. Nieman, L.B. Rogers, “Supercritical Fluid Chromatography Applied to the

Characterization of Siloxane-based Gas Chromatographic Stationary Phase”,

(1975) Separation Science, 10, 517-545

pentane + IPA or MeOH

linear pressure programming of

column inlet

silicone oil solute

condensed the mobile phase

metering valve after UV detector

(no BPR)

programmed pump pressure

concluded: small particles had problems, due to density gradients

by 1979

Everybody used the “pump” as a pressure source

a restrictor or metering valve to control flow rate

EVERYBODY BELIEVED GIDDINGS SERIES

pentane and propane remained popular mobile phase

very large particles preferred-

predicted problems with small particles

unrealistic probe molecules

Almost no progress in first 17 years

Misconceptions abound

BUT NOW

it gets REALLY Confusing!!!!

Milos Novotny, SR Springston, PA Peaden,

J.C. Fjeldsted, Milton L Lee“Capillary Supercritical Fluid Chromatography”

(1981) Analytical Chemistry 53, 407A-414A

seems to solve many problems

1.) Based on Giddings incorrect elutrophic series

2.) perceived problems W/ small particle

due to density gradients (Novotny, Rogers, others)

no pressure drop with capillary columns

3.) cap. GC exploding with invention of

fused silica and expiration of Golay’s patent

4.) ignore inconvenient facts

optimum flow is 1/25TH that w/ 5µm part.

flow increases with pressure

Pyrex tubes 200µm ID, isobaric

1st capillary Chromatogram

Capillary SFC

120 min

(1982) Analytical Chemistry 54, 1090-1093

fused silica column 50µm

pressure programming

Notice that the solutes are non-polar

commercial equipment only arrived in 1985

got better fast

First Commercial SFC1084 Based SFC ca. 1982

Dennis Gere, HP

First commercial SFC

No pressure programming

3µm particles

Independent control of:

flow,

composition, including gradients

temperature, including gradients

outlet pressure

Let the REAL science begin.

BUT nearly simultaneously.....

1982

Now there are 2 camps Each thinking the Other is Wrong

Capillary Columns• SFC extension of GC High MW, low volatility

• CO2 as polar as an alcohol

• density gradients cause loss of efficiency

• pressure programming much simpler, cheaper,

more elegant than composition programming

• packed columns are too active

• higher efficiency

• FID

Packed Columns• SFC more like HPLC, small drug like

• CO2 < polar than pentane

• use binary mixture-gradients don’t cause efficiency

losses (must control outlet pressure)

• modifiers reqd. for polar molecules

• pressure is a secondary control variable w/

modifiers

• use polar modifiers and additives

• ?

• UV, MS

Capillary columns dominated. Most thought packed columns were old news

Capillary proponents largely prevented the publication of packed column results

In reality, 2 different views of the future in 2 different fields

Pathway to the Present-Charge of the light brigade

In the 1980’s a few visionary groups saw the future of SFC as a replacement technology for HPLC

• Berger at HP- density and solvent strength of CO2, additives; elution of a wide range of polar molecules with binary and ternary mobile phases.

• Dai Games in Wales –mid-1980’s early 1990’s– SFC-MS of wide range of polar nat. products, environmental toxins, veterinary drugs.

– Shamefully, had difficulty publishing.

• Marcel Caude in Paris 1985-early 1990’s– opiates-alkiloids-other

– 1st chiral column SFC separation

• Giba Geigy in Basel- Peter Daetwyler, Klaus Anton, others-mostly late 1980’s– biggest industrial user- wide range of commercial products

• Steve Lane- mid to late 1980’s– “SFC-MS in the Pharmaceutical Industry”, in “Supercritical Fluid Chromatography, R.M. Smith, ed., RSC Chromatography Monographs The

Royal Society of Chemistry, Letchworth, Herts, UK 1988. amazingly largely ignored

• Larry Taylor- now probably most widely published in SFC

• A few others

We were ridiculed

First PUBLISHED

Chiral SFC

Chromatogram

(not the first report)

The

Golden

Application

1982 to 1992

Some Progress

Proved:

the Giddings elutrophic series was wrong (‘though some still believe it)

CO2 less polar than pentane

at constant density modifier concentration dominates retention control

solvatochromic dye measurements-modifiers increase polarity greatly

additives greatly extended range of solute polarity (column activity)

BUT:

3 competing theories about bad effects of density gradients in packed columns

it was claimed that SFC could never exceed 20,000 plates with 5µm

particles (1988) (think about sub-2µm particles today)

Omni G1205A SFC1992

Independent composition, flow, temperature, outlet pressure

Pressure programming, density programming, inverse temperature programming

UV, FID. NPD, ECD detection. Capillary or packed column

performance. Compressibility compensation.

Omni (G1205A) release 1992 Avondale

R&D team named

BillWilson

Elmer Axelson

Paul Dryden

Joe Wyan

Chris Tony

Howard Stedman

Mahmoud

Abdel-Raman

Connie Nathan

Hans Van Heist

Terry Berger

Not Shown Hans Georg Haertl

whole new level of performance

140,000 isocratic plates (220,000

demonstrated)

UV + multiple GC detectors

so much for the 20,000 plate limit

This defeated the last significant

theoretical argument against

packed column SFC

BUT...

The Achilles Heel of

G1205A

UV NOISE

>1mAU @ <10Hz

analytical relegated to

major/minor component

analysis

no QA/QC

no trace contaminants

etc.

!!!!

Then, the modern era was born

Big companies no longer invented things

They wait for a start-up to invent something,

then,

if it works, buy the start-up

1995-HP spins off Berger Instruments

Berger Instruments

diversify into areas that could be strong

despite weaknesses (like HIGH UV noise)

invented first successful semi-prep, SFC-MS

and

support equipment

analytical SFC/MS

AutoPrep

Multi-Gram II

Berger Instruments Products

not shown:

MGIII

MiniGram

Gas Delivery Systems

Petroleum analyzer

Pittcon 2010 Join The Revolution!

Using Aurora’s SFC FusionTM A5 with an Agilent 1200 SL on

1.8µm Totally Porous Particles and 2.6µm Porous Shell Particles

by: Terry A Berger

CTO

Aurora SFC Systems, Inc

3 times the speed of UHPLC

with ∆P < 250 bar

14 sec

10 chromatograms/ 6minutes15 sec for each run

21 sec to prepare for next run

∆P < 230 bar

4.6x50mm, 1.8µm Zorbax RX-Sil

30% methanol at 5ml/min, 150 bar out,

50°C

ibuprofen, ketoprofen, caffeine,

theophyline, theobromine

In an attempt to get your attention.....

50

40

30

20

10

0

0 0.5 1.0 1.5 2.0 min

1

2

3

4

5

6

7

8

9

10

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15

16

17

Kinetex 2.6µm HILIC

15%, 4.75ml/min, 150b, 50°C Phenomenex Kinetex HILIC

0.4

-1

0

1

mAU

0.8 1.20 min

Note the incredibly low noise with virtually no filtering!

Made possible by Aurora’s revolutionary pumping and

back pressure regulator design

40Hz!

Noise

The LAST Hurdle

Solved!

A method developed on a 4.6x250mm column

packed with 5µm particles, was progressively

moved to smaller particles and shorter columns.

The stationary phase was Zorbax RX-Sil (silica)

Selectivity remained essentially constant

Analysis time dropped dramatically.

Resolution degraded modestly.

none of these separations was optimized

4.6x250mm, 5µm

4.6x150mm, 3.5µm

4.6x50mm, 3.5µm

4.6x50mm, 1.8µm

5 min

1.75min

0.75min

0.35min

Going Faster w/ Smaller particles

min0 0.2 0.4 0.8 1.00.6

0

5

10

15

20

25

30

35

mAU

Atenolol

0 0.2 0.4 0.8 1.0min

0.6

0

20

40

60

80

100

mAU

Propranolol

0 0.2 0.4 0.6 0.8 1.0

0

5

10

15

20

25

mAU

min

Metoprolol

5ml/min 40% [MeOH + 0.1% TEA], 30°C, 120 bar, 4.6x260mm, 5µm Regiscell

Fusion A5 SFC Conversion Module + 1200SL HPLC

And Let US NOT Forget that

SFC is the BEST Technique for Chiral Separations, Too!

August 2012 Agilent buys Aurora

The Agilent 1260 Analytical SFC SystemThe New Standard in Performance, Cost-

efficiency, Reliability and Ease-of-Use

October 12, 2015Page 39

Agilent Confidential

Highest Analytical SFC Performance.... Ever� HPLC-like Sensitivity, Precision and Dynamic Range >10,000 for

accurate quantitation of 0.01% level impurities

Lowest Operating Cost, Green Chemistry� 10-15x lower operating costs by compatibility with standard grade CO

2

instead of liquid SFC grade C

� Lowest solvent consumption and waste generation

Ease of use and Reliability� ChemStation control, data analysis and reporting

� Agilent warranty and service quality

� Single Vendor Solution – Single Vendor Support

� Let‘s talk about Method Development & SFC/MS

Makes routine analytical SFC a reality!

104

Fig. 1. Backbone structure of bile acids.

K. Taguchi et al. / J. Chromatogr. A 1299 (2013) 103–109

acid-3 ,6 -diol-N-(2-sulphoethyl)-amide,

1992(w/ 1084)

Fig. 3. MRM chromatograms of bile acid isomeric forms using 0.2% (w/v) ammonium formate in methanol/water (95/5, v/v) (A) with 0.1% (v/v) formic acid, (B) without

formic acid. U1: unconjugates of dihydroxy bile acids, U2: unconjugates of trihydroxy bile acids, G1: glycine conjugates of dihydroxy bile acids, G2: glycine conjugates of

trihydroxy bile acids, T1: taurine conjugates of dihydroxy bile acids, T2: taurine conjugates of dihydroxy bile acids.

2013

by: Terry A Berger

SFC Solutions, Inc.

Lipidomics

with

SFC-QTOF

+and

Joe Hedrick

Jennifer VanAnda

Agilent Technologies

Little Falls, DE

A. Staby, C. Borch-Jensen, S. Balchen, J. Mollerup, “ Quantitative Analysis of Marine Oils by

Capillary SupercriticalFluid Chromatography”, (1994) Chromatographia 39 697-705

Cod Liver Oil by Capillary SFC- 1994 FID detection-120 min

complex density program; 45 Identified components; separated by mass

compounds with same carbon number but different degrees of unsaturation co-eluted.

SFC Solutions, Inc.

SFC Analysis of Cod Liver Oil-GlyceridesState-of-the-art Capillary SFC-1994

12 min

Mollerup

Capillary SFC

this work

3x100mm 1.8µm

SB C18

di-

tri-

di-

tri-

SFC Solutions, Inc.

10X Faster Separation w/ Packed Columns

0

200

400

600

800

1000

1200

1400

1600Mass(D

a)

0 1 2 3 4 5 6 7 8

Retention Time (min)

SFC Solutions, Inc.

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0Retention Time (min)

700

750

800

850

900

950

750

900

Mass, Da

2.2 2.4time, min

SFC Solutions, Inc.

3x100mm SB C18 1.8µm

2 mL/min

0 1%

0.5 1%

10 5%

20 20%

Low volume nozzle

C57H98O6

9,12 both cis-,

or both trans-

C57H92O6

6,9,12

or 9,12,15

all cis-

But What About Chromatographic Resolution?

SFC Solutions, Inc.

12

degasser

Booster

BPR

SFC

waste

modifier

trap

CO2

HPLC Pump

SFC Pump

DAD

TCC

HPLC

Waste

Autosampler

3

4

10

5

7

8

SFC Mode

figure 9.17

Agilent SFC/UHPLC Hybrid

degasser

Booster

BPR

SFC

waste

modifier

trap

CO2

HPLC Pump

SFC Pump

DAD

TCC

HPLC

Waste

Autosampler

12

3

4

10

56 7

8

HPLC mode

figure 9.18

12

3

4

56

78

1 2

8 7

3,4,5,6

SFC12 overlapped injections

HPLC10 overlapped injections

Figure 9.19

the future?

that is,

of course,

the hardest part

There have been Dozens of papers on

axial and radial

thermal gradients

starting in 1975

Mostly about HPLC and UHPLC

(causing density, viscosity, and linear velocity gradients)

and their potentially horrible effects

on efficiency/resolution

for SFC

you should largely IGNORE THEM!

Std.

LD

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 2 4 6 8 10 12 14 16

Av. Linear Velocity, mm/sec

Re

du

ce

d P

late

He

igh

t

std.

LD

3x100mm, 1.8µM RX-Sil

True UHPSFC

best case in the literature

≈83% of theoretical

true UHPSFC performance

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

0 1 2 3 4 5 6 7 8 9 10 11 12

Partition Ratio, k

Pe

ak

Fid

elity

Theory

3x100mm, 1.8µm, low dispersion plumbing

Thank You for you for listening

Do We have time for questions?