Supercritical Fluid Chromatography (SFC) Method Development Intro

Blair Berger Natural Products Laboratory

Institute of Biology, Leiden University

WHAT IS SFC?

• A separation Technique closely related to HPLC

– same hardware and software

• Dense Carbon Dioxide (CO2) as Main MP

– Almost always use a Modifier, like methanol – Methanol, Ethanol, Isopropanol, acetonitrile, etc.

– Often uses a very polar Additive like TFA or TEA – formic acid, acetic acid, trifluoroacetic acid, isopropyl amine,

– dimethylethyl amine, triethylamine (TEA), etc.

– Gradient elution is common

• Uses polar packed columns,

– like Silica, CN, HILIC, NH2, Diol, Ethylpyridine, “Premier”, Etc.

• Compatible with UV, MS, ELSD, many other Detectors

– Similar sensitivity in UV to HPLC (new)

WHAT IS SFC?

• SFC is > 3-5 times Faster than HPLC

Much Higher Diffusion Coefficients

Higher Throughput >> more samples/day

Equilibrates Much Faster

• SFC has 1/3rd to 1/5th the Pressure Drop

Much Lower Viscosity Use sub-2µm particles without the need for ultra-high pressures Can use very long columns with small Particles for High Efficiency

• SFC is Orthogonal to reversed Phase HPLC

• Lower Operating Cost

Beverage-grade CO2

CO2 Costs as little as USD$0.10/Kg

Disposal Costs are minimized

• SFC is GREEN!

Recycled CO2, Low Modifier Use

Much Less Toxic Waste

Non-flammable (get rid of the fire-marshal)

degasser

Chiller

Booster

Back

Pressure

Regulator

(BPR)

Binary pump

Thermastated Column

Compartment (oven)

SFC conversion

module

CO2

mixer

CO2

P

modifier

HP Flow

Cell

LHX RHX

Injector

Detector

Agilent Technologies 1260 SFC

CO2 MeOH

Supercritical Fluid Chromatography (SFC)

Recycled by-product

of other industrial processes

Increasing Polarity

Progressively more polar solutes require

progressively more polar mobile phases (MP) and stationary phases (SP)

SFC Offers ease of use, with additional Retention Control

Temperature (oC)

30oC 40oC 50oC 60oC

Pressure (Bar)

100 150 200 250

Modifier Concentration (%)

0 10 20 30 40 50

Flow Rate (ml/min)

1.0 2.0 3.0 4.0 5.0

Recommended Operating Ranges

Chiral separation of Warfarin Phenomenex, Lux, 3um, 3ml/min, 150bar, 30C

Rt (pk1) Rt (pk2)

16

14

12

10

8

6

4

2

0

Rete

nti

on

Tim

e (

Rt)

% Modifier

0 10 20 30 40 50

Rs (pk1:pk2)

% Modifier 0 10 20 30 40 50

16 14 12 10

8 6 4 2 0

Reso

luti

on

(R

s)

Av. Plates 9000

8000

7000

6000

5000

4000

3000

2000

Eff

icie

ncy

% Modifier 0 10 20 30 40 50

MODIFIER CONCENTRATION Effect on Retention, Resolution and Efficiency

HALF CONC DOUBLE Rt

Chiral separation of Warfarin Phenomenex, Lux, 3um, 20%, 150bar, 30C

Flow Rate Effect on Retention, Resolution and Efficiency

Rt (pk1) Rt (pk2)

NEAR LINEAR EFFECT

16

14

12

10

8

6

4

2

0

Rete

nti

on

Tim

e (

Rt)

ml/min

1.0 2.0 3.0 4.0 5.0 6.0

Rs (pk1:pk2)

ONLY MODEST EFFECT

ml/min 1.0 2.0 3.0 4.0 5.0 6.0

16 14 12 10

8 6 4 2 0

Reso

luti

on

(R

s)

Av. Plates

TRADE EFFICIENCY FOR SPEED

1.0 2.0 3.0 4.0 5.0 6.0

ml/min

9000

8000

7000

6000

5000

4000

3000

2000

Eff

icie

ncy

Av. Plates

Rs (pk1:pk2)

Rt (pk1) Rt (pk2)

10 15 20 25 30 35 40 45 50 55

9000 8000 7000

6000 5000 4000 3000 2000 1000

0 oC

oC 10 15 20 25 30 35 40 45 50 55

16 14 12 10

8 6 4 2 0

Reso

luti

on

(R

s)

Chiral separation of Warfarin Phenomenex, Lux, 3um, 3ml/min, 20%, 150bar

oC

10 15 20 25 30 35 40 45 50 55

Temperature Effect on Retention, Resolution and Efficiency

6

5

4

3

2

1

0

Rete

nti

on

Tim

e (

Rt)

Eff

icie

ncy

Little effect on Retention But can have large effects on Selectivity

Normally lower temp Produces higher efficiency

*this example is an exception*

Effect due to change on selectivity

Rt (pk1) Rt (pk2)

Rs (pk1:pk2)

Pressure (Bar) 50 100 150 200 250 300

16 14 12 10

8 6 4 2 0

Reso

luti

on

(R

s)

Chiral separation of Warfarin Phenomenex, Lux, 3um, 3ml/min, 20%, 30C

Pressure Effect on Retention, Resolution and Efficiency

Pressure (Bar)

Av. Plates

12

10

8

6

4

2

0

Rete

nti

on

Tim

e (

Rt)

Eff

icie

ncy

9000 8000 7000

6000 5000 4000 3000 2000 1000

0 50 100 150 200 250 300 Pressure (Bar)

50 100 150 200 250 300

Little effect on retention

Little effect on resolution

Little effect on efficiency

16 14 12 10

8 6 4 2 0

Reso

luti

on

(s)

Pressure

R

eso

luti

on

Re

ten

tio

n MODIFIER CONCENTRATION:

Most powerful tool in changing retention Solvent Strength is Non-Linear

function of Concentration Increasing Modifier Concentration Decreases Retention, Resolution & Efficiency FLOW RATE Modest effect on resolution Means of speeding up a separation favoring a super-optimum flow Trade Efficiency for Speed TEMPERATURE Little effect on Retention Large effect on Selectivity, changing resolution Lower T >> Better Efficiency (this ex. is an exception) PRESSURE Secondary Control Variable, Little effect on Rt, Rs or Efficiency May produce effect on Selectivity

Effi

cie

ncy

Modifier Concentration

SFC Offers ease of use, with additional Retention Control Effect of Physical Parameters Summary

Re

solu

tio

n

Effi

cie

ncy

Temperature

Re

solu

tio

n

Ret

en

tio

n

Effi

cie

ncy

Flow Rate

Ret

en

tio

n

Ret

en

tio

n

Re

solu

tio

n

Effi

cie

ncy

NO (Too Little)

Adequate Retention?

Longer column Smaller particles Change selectivity Longer retention

Increase modifier [+10%] Add 10% water Change column

Change Temperature [10°C] Increase pressure [50 Bar] Change modiifer class Change column

YES

(Too much) Decrease modifier [-10%] Decrease modifier polarity Decrease pressure [-50 bar] Increase temperature [+10oC]

A Method Development Decision Tree for Polar Solutes

FINAL METHOD

YES

NO

YES NO

YES

YES NO

Method Development Decision Tree

Additives Acids for Acids Bases for Bases

Change Column

NO

Adequate Selectivity?

Adequate Resolution?

Peaks?

Initial Conditions RECOMMENDED: 30%, 50C, 150Bar

Thanks for your attention!

Question before we go to the lab?

30 40 50 60 70 80 90

6

5

4

3

2

1

Temperature, °C

Rete

ntion T

ime, m

in

EFFECT OF PHYSICAL PARAMETERS TEMPERATURE (OC)

Little effect on Retention But

Large Effect on Selectivity