Basic principles of Countercurrent Separation

What is Countercurrent Separation (CS)?
It includes the following:
Centrifugal Countercurrent Separation (CCS)
Countercurrent Chromatography (CCC)
High-speed Countercurrent Chromatography (HSCCC)
Centrifugal Partition Chromatography (CPC)
1https://cenapt.pharm.uic.edu
C Simmler | JB Friesen | GF Pauli
CENAPT, UIC, Chicago

2
A Liquid/Liquid Separation Technique…..
UP
LP
Crude extract
2 immiscible solvents
cenapt.pharm.uic.edu Simmler | Friesen | Pauli

3
A Liquid/Liquid Separation Technique…using Centrifugal Forces
A centrifuge is used to hold one phase stationary (SP)
meanwhile the other phase (mobile) moves through,
getting mixed with the SP, then separated
throughout the tubing/column
http://pubs.acs.org/subscribe/journals/tcaw/10/i07/html/07inst.html

4
A Liquid/Liquid Separation Technique…using Centrifugal Forces
cenapt.pharm.uic.edu Simmler | Friesen | Pauli http://pubs.acs.org/subscribe/journals/tcaw/10/i07/html/07inst.html
A centrifuge is used to hold one phase stationary (SP)
meanwhile the other phase (mobile) moves through,
getting mixed with the SP, then separated
throughout the tubing/column

Liquid/Liquid Separation (LLS)
Kuhni
Extraction
Columns
Continuous
Mixer-Settler
Liquid-Liquid
Extraction
Liquid/Liquid Chromatography
A. Martin & R. Synge
Centrifugal
Countercurrent
Separation (CCCS)
Gravitational
Countercurrent
Separation (GCCS)
Countercurrent
Chromatography
(CCC)
“hydrodynamic”
Centrifugal Partition
Chromatography
(CPC)
“hydrostatic”
Droplet
Counter
Current
Chromatogr.
Craig
Counter
Current
Distribution
Kostanyan
Pulsed
Rotational
Locular
Separatory
Funnels
Countercurrent Separation (CCS)
Friesen JB, McAlpine JB, Chen SN, Pauli GF
Countercurrent Separation of Natural Products: An Update
Journal of Natural Products 78: 1765-1796 (2015)
dx.doi.org/10.1021/np501065h
Liquid/Liquid Separation Techniques…..
cenapt.pharm.uic.edu Simmler | Friesen | Pauli 5

Kuhni
Extraction
Columns
Continuous
Mixer-Settler
Liquid-Liquid
Extraction
Centrifugal
Countercurrent
Separation (CCS)
Gravitational
Countercurrent
Separation (GCS)
Countercurrent
Chromatography
(CCC)
“hydrodynamic”
Chromatography
(CPC)
“hydrostatic”
Droplet
Counter
Current
Chromatogr.
Craig
Counter
Current
Distribution
Kostanyan
Pulsed
Rotational
Locular
Separatory
Funnels
Countercurrent Separation (CS)
dx.doi.org/10.1021/np501065h

Examples of “CCC” Hydrodynamic Instruments
http://www.dynamicextractions.com/index.html#about1440
Examples of Applications
with Guy Harris

Examples of “CPC” Hydrostatic Instruments
https://www.plantaanalytica.com/technology
-centrifugal-partition-chromatography.html
Examples of Applications
with Grégoire Audo

How compounds get separated in CPC/CCC?
In HPLC: difference in affinity (column material vs. mobile phase)
In CPC/CCC: difference of solubility between two phases
The Partition Coefficient K

Partition coefficient (K)
= Concentration of analyte in one phase / Concentration of analyte in the other
shake flask (partitioning) experiment
= 4/4 = 1
= 12/36 = 1/3
K = Cupper/Clower

Separation of Compounds According to their K value
K high
K = 1
K low
stationary phase
mobile phase
K = conc. stationary/conc. mobile phase

K high
K = 1
K low
Separation of Compounds According to their K
stationary phase
mobile phase

K high
K = 1
K low
stationary phase
mobile phase

What is a biphasic solvent system in CCS?
How to choose an appropriate solvent system ?
Evaluate the overall distribution of compounds
in the UP and LP of a solvent system (K)
17cenapt.pharm.uic.edu Simmler | Friesen | Pauli

Biphasic Solvent System
= Mixture of multiple solvents that form two immiscible phases
Usually more than 2 solvents ( generally 3-4) are utilized in CCS to increase
the selectivity towards the compounds of interest.
Examples:
1 hexane : acetonitrile : methanol 10:5:5 HAcM
2 hexane : methyl t-butyl ether : acetonitrile : water 8:2:8:2 HterAcWat
3 hexane : ethyl acetate : methanol: water 5:5:5:5 HEMWat
4 chloroform : methanol : water 10:7:3 ChMWat
5 methyl t-butyl methyl ether : acetonitrile : water 4:6:10 terAcWat
6 ethyl acetate : 1-butanol : water 4:6:10 EBuWat
7 hexane : methyl t-butyl ether : acetonitrile 10:1:10 HterAc
8 dichloromethane: ethyl acetate : methanol : water 5:5:5:5 DEMWat
9 hexane : methyl t-butyl ether : methanol : water 5:5:5:5 HterMWat
Scout Solvent Systems

Diversity of Biphasic Solvent Systems
Alcohol/Ionic Aqueous or Acetonitrile/Ionic Aqueous
Aqueous Two Phase Solvent Systems (ATPS)
e.g. Polyethylene Glycol/Buffered Aqueous
Organic/Organic or “non-aqueous” e.g. Heptane/Methanol
Organic/Aqueous e.g. Hexanes/Ethyl Acetate/Methanol/Water

Where to Start? ….Which Biphasic Solvent System? ….What K?
Literature Measure K G.U.E.S.S Predict (in silico)
TLC based
Scout systems
Reported systems
LC/GC/ NMR… TLC
Structure + solvents
Shake-flask
Real partitioning
1.
2.
Liu Y, Friesen JB, McAlpine JB, Pauli GF
Solvent System Selection Strategies in Countercurrent Separation
Planta Medica 81: 1582-1591 (2015)
Brent Friesen, J. Pauli, Guido F.
“G.U.E.S.S.—A Generally Useful Estimate of Solvent Systems for CCC”
Journal of Liquid Chromatography & Related Technologies
28 (17): 2777-2806 (2005)
Laura Tyler poster #93

Where to Start? ….. Which Solvent System? ….What K?
TLC based
Scout systems
Reported systems
LC/GC/ NMR… TLC
Shake-flask
Real partitioning
1.
2.
Planta Medica 81: 1582-1591 (2015)
G.U.E.S.S.—A Generally Useful Estimate of Solvent Systems for CCC
28 (17): 2777-2806 (2005)
COSMO-RS model

Where to Start ? …. Which Solvent System?
The different organic/aqueous solvent system families
Abbreviations Solvent mixtures
HEMWat Hexanes/Ethyl Acetate/Methanol/Water
ChMWat Chloroform/Methanol/Water
EBuWat Ethyl Acetate/Butanol/Water
terAcWat Methyl t-Butyl Ether/Acetonitrile/Water
1. Perform Shake-flask experiment with the portal mixture of each solvent system family
2. Observe/measure the distribution of your compounds in UP and LP (TLC, HPLC, NMR)
More polar, glycosidated cpds
Workhorse

Working with Solvent System Families… EBuWat & terAcWat
Friesen, J Brent, and Guido F Pauli.
Rational Development of Solvent System Families in Countercurrent Chromatography.
Journal of Chromatography A 1151, no. 1–2 (June 1, 2007): 51–59.
Portal = first solvent composition to try in the considered family

Working with System Families…. HEMWat
organic/organic modifier/aqueous modifier/aqueous
+ versatile and tunable
• heptane, or pet. ether, or limonene for hexanes
• ethanol for methanol
+ good CCC/CPC performance (high Sf values)
Hexane/ Ethyl Acetate / Methanol/ Water = HEMWat
(or ARIZONA) family of solvent systems
Mcalpine, James B, J Brent Friesen, and Guido F Pauli. Natural Products Isolation.
Edited by Satyajit D. Sarker and Lutfun Nahar. Vol. 864. Methods in Molecular
Biology. Totowa, NJ: Humana Press, 2012.
Portal: working very well for first step fractionation of crude extracts!
Laura Tyler poster #93

James B McAlpine, J Brent Friesen, and Guido F Pauli. Natural Products Isolation.
Edited by Satyajit D. Sarker and Lutfun Nahar. Vol. 864. Methods in Molecular
Biology. Totowa, NJ: Humana Press, 2012.
Often modified by adding Hexanes → HChMWat
Koichi Inouie et al. “Purification of Curcumin , Demethoxycurcumin , and
Bisdemethoxycurcumin by High-Speed Countercurrent Chromatography,”
Journal of Agricultural and Food Chemistry 2008, 9328–36.
Purification of curcuminoids
from 25 mg of turmeric powder
Curcumin
K= 0.68
Bidesmethoxycurcumin
K = 1.64
Desmethoxycurcumin
K = 1.03
Working with Solvent System Families... ChMWat
Portal

Basic Principle of a Shake-Flask Experiment to
(1) measure K
(2) observe the distribution of compounds in both phases
Same volume
1. Mix 2-phase SS
2. Add sample to vial
3. Add equal amounts of upper and lower phase
4. Shake it up!
5. Separate upper and lower phase
6. Analysis of upper and lower phase
Same volume

Shake-Flask and Solvent System Selection
HEMWat 0
5/5/5/5
TerAcWat-1
4/6/10
ChMWat +4
10:7:3
Turmeric Extract
UP LP
25 mg 6 mL
6 mL
Comparison of different portal solvent systems

HEMWat
5/5/5/5
HEEWat
5/5/5/5
TerAcWat
4/6/10
ChMWat
10/7/3
HChMWat
5/10/7/3
HTerAcWat
2/4/6/10
Better solubility
Push cpds towards aqueous phase
Speed-up phase separation
Push cpds towards aqueous phase
Speed-uo phase separation

Compound distribution in the UP and LP
Spot the same volume of UP and LP
HEMWat
5/5/5/5
HEEWat
5/5/5/5
TerAcWat
4/6/10
ChMWat
10/7/3
HChMWat
5/10/7/3
HTerAcWat
2/4/6/10
HEMWat
5/5/5/5
HEEWat
5/5/5/5
TerAcWat
4/6/10
ChMWat
10/7/3
HChMWat
5/10/7/3
HTerAcWat
2/4/6/10
365 nmVisible
UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP

M 5444 E 5444 M 6464 E 6464 M 5444 E 5444 M 6464 E 6464 M 5444 E 5444 M 6464 E 6464
UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP
HEMWat Solvent System: Replacing MeOH by EtOH
Checking the overall distribution of your compounds in both phases by TLC ( same volume spotted ~ 10 µL)
Reduces overall emulsion tendency, favors a clear phase separation
HEMWat 5:4:4:4 HEEtWat 5:4:4:4
CPC for chlorophyll removal?
Seon-Beom Kim’s poster # 240

Planta Medica 81: 1582-1591 (2015)
Where to Start? ….. Which Solvent System? ….What K?
G.U.E.S.S.—A Generally Useful Estimate of Solvent Systems for CCC
28 (17): 2777-2806 (2005)
TLC based
Scout systems
Reported systems
LC/GC/ NMR… TLC
Shake-flask
Real partitioning
1.
2.

Basic Principle of G.U.E.S.S.
Vol.
K = 1
K = 0.25
K = 4
Rf = 0.5
Rf = 0.6
Rf = 0.4
sweet
spot
TLC
Generally Useful Estimation of Solvent Systems
combines the convenience of TLC with the separation power of HSCCC.
CCC
TLC elution with the UP
of your Solvent System
“G.U.E.S.S.—A Generally Useful Estimate of Solvent Systems for CCC”
28 (17): 2777-2806 (2005)

• Start with 2 immiscible solvents and add one or more modifiers
• Check the overall distribution of your compounds in both phases.
Chloroform/Water
Chloroform/Methanol/Water (ChMWat)
Hexane/Water
Hexane/Methanol/Water
Hexane/ Ethyl Acetate /Water
Hexane/ Ethyl Acetate /Methanol/Water (HEMWat)
Be Bold…..Create Your Solvent System
Mixology
Dr. Malca’s poster # 204

Question 1 – Why K?
1. Knowing the K values of your compounds helps you calculate their
retention times during your CCS.
2. Knowing the K values helps you to compare different CCS experiments,
on different instruments.
Question 2 – Really??
1. If you just want to fractionate a crude extract, you don’t need to
determine the K of your compounds.
2. In this case you can run a shake-flask experiment and simply observe the
overall distribution of compounds between LP and UP to choose your SS.
1. Why do I Need to Determine the K Value of Compounds?
2. Is it Always Necessary?
Dr. Zhou poster # 314, Dr. Malca’s poster # 204, Dr Tang’s poster # 117

Some Key Parameters when running a CCS
 Elution mode
 K vs. volume of retention
 Sf and column volume
 Rotation speed & flow rate

Parameters in CCS : Direction of elution
Choosing which phase is your stationary phase
Upper phase = Stationary phase
Reversed phase chromatography
Lower phase = Stationary phase
Normal phase chromatography
Lower phase = mobile
HEAD
TAIL
DESCENDING
Upper phase = mobile
Get mixed
Goes back up
ASCENDING

Relation between K & vol. of retention in your chromatogram
Vret Retention Volume
VSP Stationary Phase (SP) Volume in the column
VMP Mobile Phase (MP) Volume in the column
K Partition Coefficient
Vret = K X VSP + VMP
You can exactly determine in which test tubes are your compounds of interest
without hyphenated detection!
How to determine VSP and VMP

column
Stationary phase (SP)1. Fill the column with SP
2. Apply centrifugal forces
= rotation (speed in rpm)
3. Introduce the MP
at chosen flow rate
4. Equilibrium between SP/MP
When MP comes out of the coil.
SF can be calculated.
SPMP Vout
Vcolumn
VSP
VMP
OUTIN
Parameters in CCS : The stationary phase volume ratio Sf
Sf = VSP/Vcolumn
Sf = (Vcolumn –Vout)/Vcolumn
To determine VSP and VMP
10
20
30
40
50
60
70
80
90
100

Now you have your solvent system… You can start your CCS….
1. Dissolve your sample in mixture of UP/LP (generally 50%)
• Everything should be in solution, no particles
• You may want to filter your sample
• Write down – mass of sample/volume of solvent
2. Fill your CPC/CCC instrument with the phase you chose as stationary
• 2 column volumes minimum,
• Slow rotation of the column(s)
3. Start rotating your instrument at the desired speed
• Ex: 800 rpm for CCC, 2500 rpm for CPC
4. Set up your mode of elution (direction of the column rotation)
• Stationary phase = organic (UP) direction of flow = descending or “head to tail”
5. Start pumping your mobile phase into the instrument at the desired flow rate
• Ex: 1.5-2 mL/min in CCC, 25 mL/min in CPC
6. Wait for the “equilibrium” to be reached (or not, your choice)..
• to determine how much of the stationary phase remains in the column (Sf)
7. Inject your sample ….beware: do not inject air bubbles!
8. Collect your fractions!
Degas your solvent before use!
Before loading the sample,
fill the loop with your MP!

The ASP organizing committee, particularly Laura Stoll
NCCIH and ODS at NIH (U41 AT008706)
ACKNOWLEDGEMENTS

REPOSITORY
REPORTING CCS PARAMETERS
EXAMPLE OF APPLICATIONS
https://cenapt.pharm.uic.edu
41

42
Reported Parameters for Reproducibility
With a CPC instrument With an HSCCC instrumentDuration = 1 hour max Duration = 5 hours
25 mg
780 rpm
1mL/min
4.24 g2500 rpm
25 mL/min
INOUE, KOICHI, et al. Journal of Agricultural and Food Chemistry 56, no. 20 (2008): 9328–36.Simmler, C., et al. Fitoterapia 121 (2017): 6-15.
Crude extract fractionation

Countercurrent Separation as a first step for the
reproducible fractionation of crude botanical extract
CE
Fr1 GI0345 01
Fr2 GI0345 02
Fr3 GI0345 03
Fr4 GI0345 04
Fr5 GI0345 05
Fr6 GI0345 06
Fr7 GI0345 07
Fr8 GI0345 08
Fr9 GI0345 09
Fr10 GI045 10
Fr11 Gi0345 11
Glycyrrhiza inflata
Crude extract
(3x 4g injected)
HEMWat 0
3x 50 min max
½ prepHPLC
½ prepHPLC
Sf= 83%
25 mL/min, 2500 rpm
Reverse mode
K= 1.02
Precipitation: 90.9% w/w
13 mL fractions
Simmler/Lankin/Nikolić/van Breemen/Pauli Fitoterapia 121 (2017). doi:10.1016/j.fitote.2017.06.017
new
new
Licochalcone A
FRACTIONATION PURIFICATION

Crudeextract
Fr1 GI0345 01
Fr2 GI0345 02
Fr3 GI0345 03
Fr4 GI0345 04
Fr5 GI0345 05
Fr6 GI0345 06
Fr7 GI0345 07
Fr8 GI0345 08
Fr9 GI0345 09
Fr10 GI045 10
Fr11 Gi0345 11
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0
Crude extract
+
-
51.6% w/w
8.2% w/w
Mass balance
recovery : 90.8% w/w
Licochalcone A
UHPLC-UV profiles
LicA
LicA
Countercurrent Separation as a first step for the
reproducible fractionation of crude botanical extract

Basic principles of Countercurrent Separation

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