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Working with CCC solvent systems PDF Print E-mail
Written by Lucas Chadwick   
Wednesday, 28 January 2009 16:17

The solvent system in CCC is analogous to the combination of column AND mobile phase in adsorption chromatography.  The solvent system is therefore the essence of a successful CCC separation.   In general, an ideal CCC solvent system* is one in which the target compound is partitioned equally between the two phases, and has good solubility in each phase. 

The following are the steps that should be taken when choosing a CCC solvent system:

 1.  Look to the literature.  Use whatever literature database and networking services you have at your disposal.  If a CCC-based isolation method has already been developed for your target compound, do your best to find it.  That is the place to start.  Methods you discover in the literature may be based on simple "neutral" systems such as those in the table below, or may be a more elaborate method using ionic modifiers to achieve a separation based on pKa or pKb in combination with solubility (this is common for organic acids and bases, e.g. alkaloids).  Depending on your sample matrix or solvent preferences it may be advantageous to modify the solvent system found in the literature (e.g. replace chloroform with methylene chloride, MeOH with EtOH, 2-butanol with 1-butanol, heptane with hexane, etc.)

 2.  Try HEMWat.  Mixtures of Hexanes, Ethyl acetate, Methanol, and Water (HEMWat) span a large polarity range.  HEMWat systems are highlighted in the table below to illustrate the range of chemical classes that can be isolated with this popular system.  Depending on your preferences, you may substitue ethanol for methanol or heptane for hexane.  HEMWat solvent systems are generally useful for a broad polarity range of small molecules, and one of our applications specialists are standing ready to teach you how to rely on the HEMWat family for routine use in CCC in much the same way that you rely on a C18 column for routine use in HPLC.  In perfect analogy with a C18 column, when HEMWat fails it is typically because (A) the sample has very poor solubility in the system, (B) the target compound is too lipophilic or hydrophilic for the system, and/or (C) the target compound(s) happen to overlap with an impurity.  If you are experiencing case (A) or (B), skip to step 3 below.  In case of (C), it is usually worthwhile to use HEMWat for pre-concentration by running your sample at ultra-high loading in the HEMWat system, then re-running the overlapping peaks in an "orthogonal" solvent system, e.g. a system that is qualitatively altogether different, but with similar overall polarity.

3.  Pick a system from the table below. This choice can be daunting at first, but after a little experience it gets relatively easy.  First of all focus on the solubility of your sample/target.  Typically you already know what solvent(s) is(are) best at dissolving your sample or target compound.  Pick a solvent system below that contains such solvent(s).  If you have an analytical CCC instrument (Vc < ca. 20 ml), after you choose a solvent system, its typically more efficient to run the sample on the CCC right away. With a 20 ml column employing EECCC conditions on a first-pass (non-optimized) separation, it typical to consume under 30 ml solvent regardless of the injection mass.  Note that you will use more than 30 ml.  However, only about 30 ml will be "contaminated" with sample, and everything else can be recycled.**

Here is a typical first-pass workflow, in this example the CCC column volume is 18 ml.

  • Mix up (or try to always have on hand) at least 100 ml total of the solvent system.  After allowing the phases to equilibrate, separate into "UP" (upper phase) and "LP" (lower phase) pump reservoirs.  
  • Fill column with stationary phase (SP).  Generally choose whichever phase (UP or LP) you have most of to be the stationary phase.
  • Inject sample
  • Start spinning CCC and elute with mobile phase (MP) for one column volume (ca. 20 ml w/ extra-column volume).  The first ca. 8-10 ml are generally 100% recyclable SP.  Start collecting fractions as soon as the mobile phase breaks through (generally apparent as a spike on the UV detector; some investigators choose to add an unretained UV marker compound to unknown samples to ensure that the MP front is detected).
  • Extrude with SP for 1 column volume. 

If you only have a larger instrument it may be a good idea to determine the K-value (partition coefficient) of your target in the solvent system before running the sample on the CCC. If the partition coefficient is between ca. 0.25 and 4.0, its worth running your sample in the system on the CCC (and meanwhile do a few more shake-flask experiments to optimize the K-value closer to 1.0).  If your target strongly favors the upper phase (K>4) try a system higher up on the table below.  If your target strongly favors the lower phase (K>0.25), try a system lower down on the table.  If you reach the top of the table and your compound/sample still greatly favors the upper phase, or reach the bottom of the table and it still favors the lower phase, please give us a call, as we probably know how to help.

 The following table is by no means comprehensive; it is a representative list of 100 CCC solvent systems that have been successfully applied to natural products isolations, roughly in order from most lipophilic at the top to most polar at the bottom, most published in peer-reviewed journals over the last few years, with a few "oldies but goodies" thrown in.  See also Dr. J. Brent Friesen's insightful entries on TheLiquidPhase (e.g. http://theliquidphase.org/index.php?title=Literature_Statistics_2000-2007)

Target class/compound(s)

Solvent system

References

carotenes/hydrocarbons/ lycopene

hexane-CH2Cl2-MeCN (30:11:18)

Baldermann, et al. Journal of Chromatography, A 2008, 1192, 191-193.

carotenes/hydrocarbons/ lycopene

nHexane–MeCN–CH2Cl2 (20:13:7)

Wei, et al. Journal of Chromatography, A 2001, 929, 169-173.

Benzofurans/Dibenzofurans

nHeptane–CH2Cl2–MeCN (10:3:7)

Carney, et al.  Journal of Natural Products 2002, 65, 203-211.

Diterpenes /Trachylobanes/isopimaranes

nHeptane–CH2Cl2–MeCN (10:3:7)

Block, et al. Phytochemistry 2004, 65, 1165-1171.

Triterpenes/Celastrol

nHexane–EtOAc–CCl4–MeOH–H2O (1:1:8:6:1)

Wu, et al. Journal of Chromatography, A 2004, 1028, 171-174.

naphthazarins/ alkannin/shikonin derivatives/ dimeric acetylalkannins/ dimeric isovalerylalkannins

system 2: nHexane-MeCN-MeOH (8:5:2)

Assimopoulou, et al. Biomedical Chromatography 2009, 23, 182-198.

Coumarins/Inflacoumarin A

nHexane–CHCl3–MeOH–H2O (5:6:3:2)

Wang, et al. Journal of Chromatography, A 2004, 1048, 51-57.

Carotenoids/Xanthophylls/Zeaxanthin

nHexane-THF-MeOH-H2O (4:4:4:1)

Berdahl, et al. Poster Presented at the American Society of Pharmacognosy Annual Meeting, August 5-9, 2006, Arlington, VA. 2006.

beta-ionone (GUESSmix component)

nHexane–EtOAc–MeOH–H2O (9:1:9:1)

Friesen, J. B. & Pauli, G. F.  Journal of Liquid Chromatography & Related Technologies 2005, 28, 2777-2806.

Polyacetylenes/ falcarindiol

nHexane-THF-EtOH-H2O (2:2:2:1)

Berdahl, et al. Poster Presented at the American Society of Pharmacognosy Annual Meeting, August 5-9, 2006, Arlington, VA. 2006.

Quinones/Tanshinones

nHexane–EtOH–H2O (10:7:3)

Gu, et al. Journal of Chromatography, A 2004, 1057, 133-140.

Carotenoids/Xanthophylls/Lutein/Zeaxanthin

nHexane–EtOAc–EtOH–H2O (8:2:7:3)

Chen, et al. Journal of Chromatography, A 2005, 1064, 183-186.

Alkaloids/Diterpene alkaloids/ talatizamine/ isotalatizidine/ cammaconine/ more…

C6H6–CHCl3–MeOH–H2O (5:5:7:2)

Srivastava, et al. Journal of Liquid Chromatography and Related Technologies 1999, 475 229-35, 1687-1697.

carvone (GUESSmix component)

nHexane–EtOAc–MeOH–H2O (8:2:8:2)

Friesen, J. B. & Pauli, G. F.  JLCRT 2005, 28, 2777-2806.

Aryl ketenes/Phenylethanone derivative

nHeptane–EtOAc–MeOH–H2O  (4:1:4:1)

Han, et al. Journal of Chromatography, A 2004, 1022, 213-216.

Kava lactones/ desmethoxyyangonin/ dihydrokavain/ yangonin/ kavain/ dihydromethysticin/ methysticin

nHexane–acetone–MeOH–H2O (4:1:3:1)

Mikell, et al. Journal of Liquid Chromatography & Related Technologies 2003, 26, 3069-3074.

Prenylchalcones/Licochalcone A

nHexane–CHCl3–MeOH–H2O (3:12:6:4)

Wang, et al. Journal of Chromatography, A 2004, 1048, 51-57.

carvone, ionone (GUESSmix components)

hexane-MTBE-MeCN-H2O (8:2:8:2)

Friesen, J. B. & Pauli, G. F.  JLCRT 2005, 28, 2777-2806.

Phenylpropanoids/Honokiol/magnolol

nHexane–EtOAc–MeOH–H2O (5:2:5:2)

Wang, et al. Journal of Chromatography, A 2004, 1036, 171-175.

Sesquiterpenes/Artemisinin/artemisitene/arteannuin B

nHexane–EtOAc–EtOH–H2O (6:4:5:4)

Weiss, et al. Journal of Liquid Chromatography and Related Technologies 2000, 928, 909-913.

Quinones/Tanshinones

nHexane–EtOH–H2O (20:11:9)

Gu, et al. Journal of Chromatography, A 2004, 1057, 133-140.

Alkaloids/Diterpene alkaloids/ Lappaconitine/ N-deacetyllappaconitine/ oxolappaconine/ aconitine/ more…

nHexane–CH2Cl2–MeOH–H2O (15:15:24:8)

Srivastava, et al. Journal of Liquid Chromatography and Related Technologies 1999, 475 229-35, 1687-1697.

limonoids/angolensate/7-descetoxy-7-oxogedunin/deacetylgedunin/6a-acetoxygedunin/gedunin/andirobin

nHexane–EtOAc–MeOH–H2O (1:2:1.5:1)

Pereira da Silva, et al. Phytochemical Analysis 2009, 20, 77-81.

lignans/sesamin/sesamolin

petr. ether-EtOAc-MeOH-H2O (1:0.4:1:0.5)

Wang, et al. Cereal Chemistry 2009, 86, 23-25.

naphthazarins/ alkannin/shikonin derivatives

system 1: nHexane-EtOAc-EtOH-H2O (24:22:14:21)

Assimopoulou, et al. Biomedical Chromatography 2009, 23, 182-198.

Flavonoids/Prenylflavanones/prenylchalcones/xanthohumol/8-prenylnaringenin

Hexane-EtOAc–MeOH–H2O (6:4:6:4)

Chadwick, et al.  Journal of Natural Products. 2004, 67, 2024-2032.

salicylic acid (GUESSmix component)

nHexane–EtOAc–MeOH–H2O (6:4:6:4)

Friesen, J. B. & Pauli, G. F.  JLCRT 2005, 28, 2777-2806.

Alkaloids/Naphthylisoquinoline alkaloids/ ancistrocongoline A−D

CHCl3–EtOAc–MeOH–0.1M HCl (5:3:5:3)

Bringmann, et al. Journal of Natural Products 2002, 65, 1096-1101.

Coumarins/Psoralen/isopsoralen

nHexane–EtOAc–MeOH–H2O (10:7:10:8)

Wang, et al. Journal of Chromatography, A 2004, 1055, 135-140.

Quinones /Diterpene quinone/ przewaquinone A

nHexane–CCl4–MeOH–H2O (1:3:3:2)

Han, et al. Journal of Liquid Chromatography & Related Technologies 2003, 26, 1267-1274.

capsaicinoids/dihydrocapsaicin/capsaicin/nordihydrocapsaicin

CCl4-MeOH-H2O (4:3:2)

Li, et al. Separation and Purification Technology 2009, 64, 304-308.

reserpine (GUESSmix component)

nHexane–EtOAc–MeOH–H2O (7:3:5:5)

Friesen, J. B. & Pauli, G. F.  JLCRT 2005, 28, 2777-2806.

coumarin (GUESSmix component)

nHexane–EtOAc–MeOH–H2O (6:4:5:5)

Friesen, J. B. & Pauli, G. F.  JLCRT 2005, 28, 2777-2806.

estradiol (GUESSmix component)

nHexane–EtOAc–MeOH–H2O (6:4:5:5)

Friesen, J. B. & Pauli, G. F.  JLCRT 2005, 28, 2777-2806.

anthraquinones/ aurantio-obtusin/ obtusin/ 1-desmethylchryso-obtusin

nHexane–EtOAc–MeOH–H2O (11:9:10:10)

Zhu, et al. Separation and Purification Technology 2008, 63, 665-669.

diterpenes/pseudolaric acid A & B

nHexane–EtOAc–MeOH–H2O (1:1:1:1)

Han, et al. Journal of Separation Science 2009, 32, 309-313.

furanocoumarins/psoralen/bergapten

nHexane–EtOAc–MeOH–H2O (1:1:1:1)

Chi, et al. Journal of Liquid Chromatography & Related Technologies 2009, 32, 136-143.

flavonols/ isorhamnetin

two step: nHexane–EtOAc–MeOH–H2O (5:5:5:5)-->(5:5:6:4)

Cao, et al. Journal of Liquid Chromatography & Related Technologies 2009, 32, 273-280.

quercetin (GUESSmix component)

nHexane–EtOAc–MeOH–H2O (5:5:5:5)

Friesen, J. B. & Pauli, G. F.  JLCRT 2005, 28, 2777-2806.

naringenin (GUESSmix component)

nHexane–EtOAc–MeOH–H2O (4:6:5:5)

Friesen, J. B. & Pauli, G. F.  JLCRT 2005, 28, 2777-2806.

vanillin (GUESSmix component)

nHexane–EtOAc–MeOH–H2O (3:7:5:5)

Friesen, J. B. & Pauli, G. F.  JLCRT 2005, 28, 2777-2806.

aspirin (GUESSmix component)

nHexane–EtOAc–MeOH–H2O (3:7:5:5)

Friesen, J. B. & Pauli, G. F.  JLCRT 2005, 28, 2777-2806.

umbelliferone (GUESSmix component)

nHexane–EtOAc–MeOH–H2O (3:7:5:5)

Friesen, J. B. & Pauli, G. F.  JLCRT 2005, 28, 2777-2806.

Flavonoid glycosides/ epimedokoreanoside I/ icariin/ icariside II

CHCl3–MeOH–H2O (8:7:4)

Liu, et al. Journal of Chromatography, A 2005, 1064, 53-57.

carbon nanotubes

CHCl3–MeOH–H2O (4:4:2)

Cai, et al. Journal of Liquid Chromatography & Related Technologies 2009, 32, 399-406.

macrolide antibiotics/ amphotericin B

H2O-DMSO-THF (4:4:11)

Foucault, et al. Analytical Chemistry 1993, 65, 2150-2154.

Coumarins/Psoralen/isopsoralen

nHexane–EtOAc–MeOH–H2O (10:10:9:11)

Liu, et al. Journal of Chromatography, A 2004, 1057, 225-228.

Glucosinolates/ glucoraphanin

nPrOH–MeCN–(NH4)2SO4–H2O (10:5:12:10)

Fahey, et al. Journal of Chromatography, A 2003, 996, 85-93.

Flavonoids/Flavones/chalcones/2',3,4,4'-tetrahydroxychalcone/5,6,7,4'-tetrahydroxyflavone/ butin

1st step:  CHCl3–CH2Cl2–MeOH–H2O (2:2:3:2)

Tian, et al. Journal of Chromatography, A 2004, 1049, 219-222.

Flavonoids/Flavones/chalcones/biflavonoid/quercetin-3-O-alpha-L-arabinoside/  quercetin-3-O-beta-D-galactoside/ amentoflavone

EtOAc–nPrOH–H2O (140:8:80)

Sannomiya, et al. Journal of Chromatography, A 2004, 1035, 47-51.

ferulic acid (GUESSmix component)

nHexane–EtOAc–MeOH–H2O (3:7:4:6)

Friesen, J. B. & Pauli, G. F.  JLCRT 2005, 28, 2777-2806.

reserpine, ferulic acid, aspirin, vanillin, umbelliferone, quercetin, coumarin (GUESSmix components)

hexane-MTBE-MeCN-H2O (4:6:4:6)

Friesen, J. B. & Pauli, G. F.  JLCRT 2005, 28, 2777-2806.

diterpene glycosides/glucopyranosides of pseudolaric acid A & B

nHexane–EtOAc–MeOH–H2O (1:9:4:6)

Han, et al. Journal of Separation Science 2009, 32, 309-313.

Flavone-C-glycosides/ apigenin 6-C-galactosyl-6''-O-b-galactopyranoside/ isoorientin/ coeluters: orientin & isovitexin

EtOAc–nBuOH–MeOH–H2O (35:10:11:44)

Oliveira, et al. Phytochemical Analysis 2003, 14, 96-99.

Tetramethylpyrazines/Chuanxiongzine

nHexane–EtOAc–EtOH–H2O (5:5:3:7)

Li, et al. Journal of Chromatography, A 2004, 1047, 249-253.

Alkaloids/Aporphine alkaloids/ neolitsine/ dicentrine/ cassythine/ actinodaphnine

CH2Cl2–MeOH–5% HOAc (5:5:3)

Stevigny, et al. Planta Medica 2002, 68, 1042-1044.

Diterpenes/10-Deacetylbaccatin III

2nd step: nHexane–CHCl3–MeOH–H2O (5:25:34:20)

Cao, et al. Journal of Chromatography, A 1998, 21, 397-401.

Flavonoids/Flavones/chalcones/2',3,4,4'-tetrahydroxychalcone/5,6,7,4'-tetrahydroxyflavone/ butin

2nd step:  CH2Cl2–MeOH–MeCN–H2O (40:11:25:20)

Tian, et al. Journal of Chromatography, A 2004, 1049, 219-222.

Diterpenes/10-Deacetylbaccatin III

1st step: nHexane–EtOAc–EtOH–H2O (2:5:2:5)

Cao, et al. Journal of Chromatography, A 1998, 21, 397-401.

Saponins/ Ginsenosides/ ginsenoside-Rg1/ ginsenoside-Rd/ notoginsenoside-R1/ ginsenoside-Re/ ginsenoside-Rb1

1st step:  CHCl3–MeOH–2-BuOH–H2O (5:6:1:4)

Cao, et al. Journal of Liquid Chromatography & Related Technologies 2003, 26, 1579-1591.

Monoterpenes/Iridoid glycosides / catalpol glycosides

CHCl3–MeOH–nPrOH–H2O (5:6:1:4)

Helfrich, E. & Rimpler, H. Phytochemistry 2000, 54, 191-199.

Saponins/Phytolacca saponins

CHCl3–MeOH–IPA–H2O (5:6:1:4)

Hostettmann, et al. Journal of Liquid Chromatography and Related Technologies 2001, 24, 1711-1721.

saponins/ platycodin D3 & platycodin D & their deapiose forms

Hexane–butanol–H2O (1:10:5)

Ha, et al. Phytochem Anal 2009, 20, 207-213

Flavonolignans/ silymarin / silychristin/ silybin/ isosilybin

nHexane–EtOAc–MeOH–H2O (1:4:3:4)

Du, et al. Journal of Liquid Chromatography & Related Technologies 2002, 25, 2515-2520.

Tea Catechins (Camelia sinensis catechins) / EGCG/ ECG/ EAG

nHexane–EtOAc–MeOH–H2O (3:10:3:10)

Degenhardt, et al. Journal of Agricultural and Food Chemistry 2000, 925, 3425-3430.

chlorogenic acid, reserpine, aspirin (GUESSmix components)

hexane-MTBE-MeCN-H2O (2:8:2:8)

Friesen, J. B. & Pauli, G. F.  J. Chrom. A. 2007, 1151, 51-59.

Cyclodepsipeptides/ petrosifungin A & B

n-Heptane–EtOAc–MeOH–H2O (2:8:2:8)

Bringmann, et al. Journal of Natural Products 2004, 67, 311-315.

saponins/ platycoside E/ deapio-platycoside E

hexane–butanol–H2O (1:40:20)

Ha, et al. Phytochem Anal 2009, 20, 207-213

Proanthocyanidins

nHexane–EtOAc–MeOH–H2O (1:5:1:5)

Degenhardt, et al. Journal of Agricultural and Food Chemistry 2000, 925, 3425-3430.

Flavonoid glycosides/ baicalein/ baicalein-7-o-glucoside/ baicalein-7-O-diglucoside/ chrysin

nHexane–EtOAc–MeOH–H2O (1:8:1:8)

Chen, et al. Journal of Chromatography, A 2005, 1063, 241-245.

Lignans/Arctiin

EtOAc–nBuOH–EtOH–H2O (10:1:2:10)

Wang, et al. Journal of Chromatography, A 2005, 1063, 247-251.

tannic acid (GUESSmix component)

nHexane–EtOAc–MeOH–H2O (1:9:1:9)

Friesen, J. B. & Pauli, G. F.  J. Chrom. A. 2007, 1151, 51-59.

Phenylpropanoid glycosides/Verbascoside

EtOAc–MeCN–H2O (13:13:24)

Viron, et al. Phytochemical Analysis 1998, 335, 39-43.

Saponins/Glycyrrhizin

EtOAc–MeOH–H2O (5:2:5)

Jiang, et al. Journal of Chromatography, A 2004, 1033, 183-186.

Isoflavone glycosides/ calycosin-7-O-beta-D-glycoside/ formononetin-7-O-beta-D-glycoside

2nd step:  EtOAc-EtOH-H2O (5:1:5)

Ma, et al. Journal of Chromatography, A 2003, 992, 193-197.

Alkaloids/Benzylisoquinoline alkaloids/hydrastine/canadine/canadaline

CHCl3-H2O (1:1), 40mM HCl in UP; 40mM TEA in LP

Chadwick, et al. Journal of Liquid Chromatography and Related Technologies 2001, 348, 2445-2453.

procyanidins/ procyanidin B1/ procyanidin B2/ procyanidin A2/ procyanidin C1

EtOAc-IPA-H2O (20:1:20)

Koehler, et al. Journal of Chromatography A. 2008, 1177, 114-125.

Catechins/Epigallocatechin

nHexane–EtOAc–H2O (1:9:10)

Cao, X. & Ito, Y.  Journal of Liquid Chromatography & Related Technologies 2004, 27, 145-152.

Flavonoid glycosides/ 5,7-dihydroxy-3',4'-trihydroxyflavone-3-O-6''-rhamnoside/ 5,7-dihydroxy-3',4'-dihydroxyflavone-3-O-6''-rhamnoside

nHexane–EtOAc–MeOH–H2O (2:12:3:15)

Du, et al. Journal of Chromatography, A 2004, 1040, 147-149.

Phenylpropanoid glycosides /Acteoside

EtOAc–H2O (1:1)

Li, et al. Journal of Chromatography, A 2005, 1063, 161-169.

caffeine, chlorogenic acid (GUESSmix components)

EtOAc-H2O (1:1)

Friesen, J. B. & Pauli, G. F.  Journal of Chromatography A. 2007, 1151, 51-59.

procyanidins/ procyanidin B1/ procyanidin B2/ procyanidin A2/ procyanidin C1

EtOAc-BuOH-H2O (14:1:15)

Koehler, et al. Journal of Chromatography A. 2008, 1177, 114-125.

Isoflavan glycosides/ pterocarpan glycosides/ dihydroxy-3',4'-dimethyl isoflavan-7-O-beta-D-glucopyranoside /di-methoxypterocarpan-3-O-beta-D-glucopyranoside

EtOAc–EtOH–HOAc–H2O (16:4:1:20 )

Ma, et al. Journal of Chromatography, A 2004, 1023, 311-315.

nicotinic acid, caffeine, chlorogenic acid (GUESSmix components)

EtOAc-nButanol-H2O (8:2:10)

Friesen, J. B. & Pauli, G. F.  J. Chrom. A. 2007, 1151, 51-59.

nicotinic acid, chlorogenic acid, reserpine (GUESSmix components)

MTBE-MeCN-H2O (8:2:10)

Friesen, J. B. & Pauli, G. F.  J. Chrom. A. 2007, 1151, 51-59.

Flavonol glycosides/quercetin-3-O-beta-D-glucoside/isorhamnetin-3-O-beta-D-glucoside/syringetin-3-O-beta-D-glucoside
isorhamnetin-3-O- -d-glucoside

n-hexane–nBuOH–H2O (1:1:2)

Gutzeit, et al. J. Chrom. A, 1172 (2007) 40–46

Phenolic glycosides/Gastrodin

EtOAc–nBuOH–H2O (3:2:5)

Li, H.-B. & Chen, F. Journal of Chromatography, A 2004, 1052, 229-232.

nicotinic acid, caffeine, chlorogenic acid (GUESSmix components)

EtOAc-nButanol-H2O (6:4:10)

Friesen, J. B. & Pauli, G. F.  J. Chrom. A. 2007, 1151, 51-59.

Saponins/Ginsenosides/ ginsenoside-Rb1/ notoginsenoside-R1/ ginsenoside-Re/ ginsenoside-Rg1

nHexane–nBuOH–H2O (3:4:7)

Du, et al. Journal of Chromatography, A 2003, 1008, 173-180.

Saponins/ Ginsenosides/ ginsenoside-Rg1/ ginsenoside-Rd/ notoginsenoside-R1/ ginsenoside-Re/ ginsenoside-Rb2

2nd step:  EtOAc–nBuOH–H2O (1:1:2)

Cao, et al. Journal of Liquid Chromatography & Related Technologies 2003, 26, 1579-1591.

L-tryptophan, salicin, p-arbutin, nicotinic acid, caffeine, chlorogenic acid (GUESSmix components)

EtOAc-nButanol-H2O (4:6:10)

Friesen, J. B. & Pauli, G. F.  J. Chrom. A. 2007, 1151, 51-59.

Bilberry/Blueberry anthocyanins/ delphinidin-3-O-sambubioside/ cyanidin-3-O-sambubioside

MTBE–nBuOH–MeCN–H2O–TFA (1:4:1:5:0.01)

Du, et al. Journal of Chromatography, A 2004, 1045, 59-63.

Isoflavone glycosides/ calycosin-7-O-beta-D-glycoside/ formononetin-7-O-beta-D-glycoside

1st step:  EtOAc–nBuOH–EtOH–H2O (30:10:6:50)

Ma, et al. Journal of Chromatography, A 2003, 992, 193-197.

Flavone-C-glycosides /Icariin

nHexane–nBuOH–MeOH–H2O (1:4:2:6)

Du, et al. Journal of Chromatography, A 2002, 962, 239-241.

Hydrolysable tannins/Ellagic acid / corilagin

nBuOH–HOAc–H2O (4:1:5)

Jikai, et al. Phytochemical Analysis 2002, 13, 1-3.

Anthocyanins/ delphinidin 3-rutinoside/ cyanidin 3-rutinoside/
delphinidin 3-glucoside/ cyanidin 3-glucoside

MTBE–nBuOH–MeCN–H2O (2:2:1:5)

Degenhardt, et al. Journal of Agricultural and Food Chemistry 2000, 48, 338-343.

Flavonol glycosides/ baicalin

nBuOH–H2O (1:1)

Lu, et al. Journal of Chromatography, A 2003, 1017, 117-123.

labile pigments/ tunichromes/ tunichrome B-1

iAmOH-nBuOH-nPrOH-H2O-HCOOH-tbutyl sulfide (32:48:40: 120:1:4)

Bruening, et al. Journal of Natural Products 1986, 49, 193-204.

Lignan glycosides/ secoisolariciresinol diglucoside

MTBE–nBuOH–MeCN–H2O (1:3:1:5)

Degenhardt, et al. Journal of Chromatography, A 2002, 855, 299-302.

caffeine, nicotinic acid, chlorogenic acid, reserpine (GUESSmix components)

MTBE-MeCN-H2O (5:5:10)

Friesen, J. B. & Pauli, G. F.  J. Chrom. A. 2007, 1151, 51-59.

p-arbutin, salicin, caffeine, nicotinic acid, chlorogenic acid, reserpine, vanillin (GUESSmix components)

MTBE-MeCN-H2O (4:6:10)

Friesen, J. B. & Pauli, G. F.  J. Chrom. A. 2007, 1151, 51-59.

oxygenated monoterpenes/ 2,6-dimethyl-2,5-heptadienoic acid & its β-glucopyranosyl ester, 2,6-dimethyl-5,7-octadiene-2,3-diol/ 3,7-dimethyl-3-octene-1,2,6,7-tetrol

CHCl3–MeOH–H2O (7:3:18)

Osorio, et al. Phytochemistry 2000, 53, 97-101.

2-BuOH - 2-butanol or isobutanol
C6H6 - benzene
CCl4 - carbon tetrachloride
CH2Cl2 - methylene chloride or dichloromethane
CHCl3 - chloroform
DMSO - dimethylsulfoxide
EtOAc - ethyl acetate
EtOH - ethanol
H2O - water
HOAc - acetic acid
iAmOH - iso-amyl alcohol
IPA - isopropanol or 2-propanol
MeCN - acetonitrile
MeOH - methanol
MtBE - methyl-t-butyl-ether
nBuOH - n-butanol or 1-butanol
nPrOH - 1-propanol or n-propanol
THF - tetrahydrofuran

*This page is dedicated largely to "neutral" solvent systems wherein the mechanism of separation is differential partitioning between two immiscible phases.  Check back soon for information about techniques for isolating ionizable compounds such as  (e.g. pH-zone refining CCC, pH peak focusing CCC, and strong ion displacement CCC).  It is also worth noting that a majority of the solvent systems described in this article were developed for isolation of small molecule (< ca. 2000 D) natural products.  Check back soon for articles focused on macromolecules such as proteins, RNA, and DNA.

**A few notes on laboratory solvent conservation:  Any time the instrument will sit unused for more than a few days, fill it with 25% MeOH (aq.) using the lowest-cost MeOH in your lab (its a good idea to filter it first).   When you are ready to fractionate a sample, blow out the aqueous MeOH with air or nitrogen.  This allows you to recycle all but the first few ml of SP that elute from the outlet. Using the 20 ml instrument using the above EECCC workflow (elute for 1 column volume; extrude for 1 column volume) as an example, it is generally possible fractionate a sample (up to over 200 mg demonstrated in our lab on an 18 ml instrument) into fractions totaling about 30 ml in volume, for an average fraction concentration of up to over 7 mg/ml.  The remainder of the solvent system can be immediately separated back into the UP and LP reservoirs and reused.   

 
Last Updated on Tuesday, 14 July 2009 19:38