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)
 Supercritical Fluid Chromatography (SFC)

degasser
SFC conversion
module CO
CO22 MeOH
modifier

Booster
Binary pump
P mixer

Chiller
Agilent Technologies
1260 SFC
Back Injector
Pressure
Regulator
(BPR)

LHX RHX Thermastated Column

CO2 Compartment (oven)
Recycled by-product
of other
industrial processes
HP Flow
Cell Detector
 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

Modifier Concentration (%) Temperature (oC)

0 10 20 30 40 50 30oC 40oC 50oC 60oC

Flow Rate (ml/min) Pressure (Bar)

1.0 2.0 3.0 4.0 5.0 100 150 200 250

Recommended Operating Ranges

 MODIFIER CONCENTRATION
Effect on Retention, Resolution and Efficiency
16
Rs (pk1:pk2)
14
16 12
Rt (pk1) Rt (pk2) 10
14 8
6
12 4

Resolution (Rs)
2
10 HALF CONC 0
DOUBLE Rt 0 10 20 30 40 50
% Modifier
8
9000
Av. Plates
6 8000
7000

Retention Time (Rt)

4
6000

2 5000
Efficiency

4000
0 3000
0 10 20 30 40 50
% Modifier 2000
Chiral separation of Warfarin 0 10 20 30 40 50
Phenomenex, Lux, 3um, 3ml/min, 150bar, 30C % Modifier
 Flow Rate
Effect on Retention, Resolution and Efficiency
16
Rs (pk1:pk2)
16 14
Rt (pk1) 12
Rt (pk2)
10
14
8 ONLY MODEST EFFECT
6
12
4

Resolution (Rs)
2
10 0
NEAR LINEAR EFFECT 1.0 2.0 3.0 4.0 5.0 6.0
8 ml/min
9000
Av. Plates
6 8000
7000

Retention Time (Rt)

4
6000
2 5000
Efficiency

4000
0 TRADE EFFICIENCY
1.0 2.0 3.0 4.0 5.0 6.0 3000
FOR SPEED
ml/min 2000
Chiral separation of Warfarin 1.0 2.0 3.0 4.0 5.0 6.0
Phenomenex, Lux, 3um, 20%, 150bar, 30C ml/min
 Temperature
Effect on Retention, Resolution and Efficiency
16
Rs (pk1:pk2)
14
6 12
Rt (pk1) Rt (pk2) 10
8
5
6
4

Resolution (Rs)
2 Effect due to change on selectivity
4
0
10 15 20 25 30 35 40 45 50 55
oC
3
9000 Av. Plates
8000
2
7000
Little effect on Retention

Retention Time (Rt)

6000
But can have large effects on Selectivity
1 5000
4000 Normally lower temp
Efficiency

3000 Produces higher efficiency

0
2000 *this example is an exception*
10 15 20 25 30 35 40 45 50 55 1000
oC
0 10 15 20 25 30 35 40 45 50 55
Chiral separation of Warfarin
Phenomenex, Lux, 3um, 3ml/min, 20%, 150bar oC
 Pressure
Effect on Retention, Resolution and Efficiency
16
Rs (pk1:pk2)
14
Rt (pk1) Rt (pk2) 12
10
12 8
6 Little effect on resolution
10 4

Resolution (Rs)
2
Little effect on retention 0
8 50 100 150 200 250 300
Pressure (Bar)
6 9000 Av. Plates
8000
7000
4

Retention Time (Rt)

6000
5000
2 4000 Little effect on efficiency
Efficiency

3000
0 2000
50 100 150 200 250 300 1000
Pressure (Bar) 0 50
Chiral separation of Warfarin 100 150 200 250 300
Phenomenex, Lux, 3um, 3ml/min, 20%, 30C Pressure (Bar)
 SFC Offers ease of use, with additional Retention Control
Effect of Physical Parameters Summary
MODIFIER CONCENTRATION:
Most powerful tool in changing retention
16 Solvent Strength is Non-Linear
function of Concentration
14

Efficiency

Retention
Increasing Modifier Concentration

Resolution
12 Decreases Retention, Resolution & Efficiency
10 Modifier Concentration
8
6 FLOW RATE
Modest effect on resolution
4 Means of speeding up a separation
2 favoring a super-optimum flow

Efficiency

Retention
Resolution(s)
0 Trade Efficiency for Speed
Flow Rate
TEMPERATURE
Little effect on Retention
Large effect on Selectivity, changing resolution
Lower T >> Better Efficiency

Resolution Resolution

Retention
Efficiency
(this ex. is an exception)
Temperature
PRESSURE
Secondary Control Variable,
Little effect on Rt, Rs or Efficiency
Efficiency

May produce effect on Selectivity

Retention
Resolution
Pressure
 A Method Development Decision Tree for Polar Solutes
Method Development Decision Tree
RECOMMENDED: 30%, 50C, 150Bar
Initial Conditions

S Peaks? Additives
NO Acids for Acids
YES Bases for Bases
Change Column
Adequate
Decrease modifier [-10%] Retention?
(Too Little) NO NO (Too much) Increase modifier [+10%]
Decrease modifier polarity Add 10% water
Decrease pressure [-50 bar] YES
Change column
Increase temperature [+10oC]
Adequate
Selectivity? Change Temperature [10°C]
YES NO Increase pressure [50 Bar]
Change modiifer class
Change column
Adequate
Resolution? Longer column
YES NO Smaller particles
Change selectivity
FINAL Longer retention

METHOD
Thanks for your attention!
Question before we go to the lab?
EFFECT OF PHYSICAL PARAMETERS
TEMPERATURE (OC)
Little effect on Retention But
6
Large Effect on Selectivity

Retention Time, min

2

1
30 40 50 60 70 80 90
Temperature, °C