The different types of instruments are compared in terms of capacity (column volume), resolution, and duration required for the separation of targeted compounds. This workshop presentation was prepared by Dr. Friesen (http://www.dom.edu/departments/physicalsciences/faculty/j-brent-friesen).

1. CCS Instrument Comparison
https://upload.wikimedia.org/wikipedia/commons/3/39/Music_wall.jpg

2. 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

3. CCC vs CPC
Berthod 2007 J of Liq Chromatog & Relat Technol volume 30 p.447

4. Survey Results
instrument volume articles
TBE-300A, TBE-
300B 260-300 105
TBE-20A 16 2
TBE-1000A 1000 10
TBE-5000A 5000 1
GS-20A2 40 11
GS-10AB, GS10A-
2, GS10A 230-240 54
Spectrum 15
mini 9
midi 15
maxi 2
FCPC
50, 200, 1000,
5000 30
CCC-1000 300-325, 850 29
PC-Inc. 8
instrument volume articles
HPCPC LLB-M 8
Sanki CPC
80, 240, 1400,
5400 6
Custom 400 - 15000 20
DCCC 3
Armen TC TC-250 1
SPOT CPC 100 light 2
HHS-400A ? 3
custom CPC 1
EMC-500A ? 1
IFBE custom 420 7
Quattro 4
D400, D1200 ? 2
SRCCC 1
Friesen 2015 Journal of Natural Products vol 78 p. 1764

5. Column Volumes
24
34
78
104
17
12
49
13
16-100
108-200
220-250
260-300
320-400
420-500
600-1000
1200-22000
Column Volumes: All CS Instruments
number of instruments
columnvolumes(mL)
Friesen 2015 Journal of Natural Products vol 78 p. 1764

6. GUESSmix in hexane / ethyl acetate / methanol / water 4:6:4:6
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.29 2.67 3.2 4 5.33 8 16 ¥K'(2)
A
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.29 2.67 3.2 4 5.33 8 16 ¥K'(2)
A
280nm
230nm
J-type centrifuge 120 mL
Fast Centrifugal Partition Chromatography (FCPC) 200 mL
220 mg
440 mg
G
H
X
T
r
C
D F
R
U
V
A
Q
M
N
Z
E O
I
Y
b

7. Figure 4. CCC and CPC chromatograms obtained by using CUAE/ CCC/CPC system.
(a) CCC chromatogram using solvent system of HEMWat at a volume ratio of 4:3:4:3;
(b) (b) CPC chromatogram using solvent system of HEMWat at a volume ratio of 4:3:4:3;
Development of Circulating Ultrasounic-Assisted Online Extraction Coupled to Countercurrent Chromatography and Centrifugal Partition
Chromatography for Simultaneous Extraction and Isolation of Phytochemicals: Application to Ligusticum chuanxiong Hort Yuchi Zhang, Chunming Liu,
Yanjuan Qi, Yuchun Li, and Sainan Li Ind. Eng. Chem. Res., 2015, 54 (11), pp 3009–3017 DOI: 10.1021/ie504179r
TAUTO TBE−300B
SIC CPC-240 (Kyoto, Japan)
instrument that was modified in our laboratory
Comparison of HSCCC & CPC

8. Figure 4. CCC and CPC chromatograms obtained by using CUAE/ CCC/CPC system.
(c) CCC chromatogram using solvent system of HEMWat at a volume ratio of 4:3:4:2; and
(d) CPC chromatogram using solvent system of HEMWat at a volume ratio of 4:3:4:2.
Development of Circulating Ultrasounic-Assisted Online Extraction Coupled to Countercurrent Chromatography and Centrifugal Partition
Chromatography for Simultaneous Extraction and Isolation of Phytochemicals: Application to Ligusticum chuanxiong Hort Yuchi Zhang, Chunming Liu,
Yanjuan Qi, Yuchun Li, and Sainan Li Ind. Eng. Chem. Res., 2015, 54 (11), pp 3009–3017 DOI: 10.1021/ie504179r
Comparison of HSCCC & CPC

9. Fig. 4. CCC, CPC of UADE extract
of white peony roots and fractions.
(a) CCC chromatogram and (b)
CPC chromatogram obtained using
the EBuEtWat (1:3.5:2:4.5, v/v/v/v)
solvent system. (c) UHPLC
chromatogram of the UADE extract
of white peony roots. (1) Albiflorin,
(2) benzoylpaeoniflorin, (3)
paeoniflorin, and (4)
galloylpaeoniflorin.
Ultrason Sonochem. 2015 26:111-118. doi: 10.1016/j.ultsonch.2015.02.017. Development of "ultrasound-assisted dynamic extraction"
and its combination with CCC and CPC for simultaneous extraction and isolation of phytochemicals. Zhang Y, Liu C, Li J, Qi Y, Li Y, Li S.
Comparison of HSCCC & CPC

10. Fig. 1. Solid line represents reversed-phase separation of standards using Pharma-Tech CCC-1000, 320mL column volume, Sf
= 68%, HEMWat 4:6:4:6 1.5m L/min, loading 1–5 mg of each 12 standards (Aspirin (A), caffeine (C), nicotinic acid (D), ferulic
acid (F), red new Coccine (R), umbelliferone (U), vanillin (V)). Dashed line is the separation of the same mixture using DE
Spectrum semi-preparative coils at 54×g (751 rpm), 136mL column volume, lower layer as mobile phase, 1.5mL/min, head to
tail elution, Sf =73 %, 60mg total loading (5mg of each 12 standards as above), monitored by DAD 254 nm, compared
separation time = 130 min.
Journal of Chromatography A, 1216 (2009) 4181–4186 Performance comparison using the GUESS mixture to evaluate counter-current chromatography
instruments. Hacer Guzlek, Philip LeslieWood, Lee Janaway
Comparison of HSCCC & HPCCC

11. Fig. 4. Separation of caffeine and vanillin with the Arizona L heptane–ethyl acetate–methanol–water 2:3:2:3 (v/v) liquid system. Reversed-phase mode: organic upper
stationary phase and aqueous lower mobile phase flown in the head to tail direction. Top chromatograms: column HPCCC Mini 19.6mL with a 0.8mm I.D. tubing coil,
2100 rpm, 2 mL/min. Sf = 38%, Rs = 2.0. Bottom chromatograms: column HPCCC Mini 20.8mL with a 1.6mm I.D. tubing coil, 1800 rpm, 5mL/min, Sf = 66%, Rs = 2.0.
Injectionvolume 0.5 mL, concentration 1mg/mL in lower mobile phase. Detection UV at 254 nm. Left chromatograms shown in volume units. Right chromatograms
shown in time units.
Berthod 2009 Journal of Chromatog A vol 1216 p 4169
HPCCC
Mini 19.6mL
0.8 mm tubing
HPCCC
Mini 20.8mL
1.6 mm tubing
Comparison of HPCCC designs

12. Fig. 5. Separation of nine GUESS solutes with HepEMWat 1:1:1:1 Organic upper stationary phase. Top chromatograms obtained in 22min:
column HPCCC Mini 19.6mL with a 0.8mmI.D. tubing coil, 2100 rpm, 2 mL/min. Sf = 46%. Bottom chromatograms
obtained in 13min: column HPCCC Mini 20.8mL with a 1.6mm I.D. tubing coil,1800 rpm, 5 mL/min, Sf = 72%. Injection volume 0.5 mL,
concentration ∼1mg/mL (each) in lower mobile phase. Detection UV at 254 nm. The inset chromatograms are
enlargements of the early eluting peaks.. Carvone (O) retention time is indicated.
HPCCC Mini 19.6mL with
0.8mmI.D. tubing coil
HPCCC Mini 20.8mL
1.6mm I.D. tubing
Berthod 2009 Journal of Chromatog A vol 1216 p 4169
Comparison of HPCCC designs