Hydrophobic and hydrophilic interactions in countercurrent chromatography

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Hydrophobic and hydrophilic interactions in countercurrent chromatographyR Lihong Zhang, Shihua Wu∗ Research Center of Siyuan Natural Pharmacy and Biotoxicology, College of Life Sciences, Zhejiang University, Hangzhou 310058, PR China

a r t i c l e

i n f o

Article history: Received 9 January 2019 Revised 20 June 2019 Accepted 26 September 2019 Available online xxx Keywords: Countercurrent chromatography Hydrophobic interactions Hydrophilic interactions Additives Ionic liquids Solvent systems selection

a b s t r a c t Countercurrent chromatography (or counter-current chromatography, CCC) is a unique support-free liquid-liquid partition chromatography. Since it was invented by Y. Ito in 1960s, CCC has been widely accepted and applied as popular separation and purification technique for natural and synthetic complex. However, up to date there is little study to address on hydrophobic and hydrophilic interactions in CCC process, although hydrophobic interaction chromatography (HIC) and hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC) as solid-support chromatographic techniques are widely applied at different stages of downstream processing. In fact, hydrophobic and hydrophilic interactions might be more popular in CCC separation than that in the liquid chromatography. For example, adding small solvents or additives in two-phase solvent systems may change significantly hydrophobic or hydrophilic interactions between solvents and solutes. Normally, CCC separation employs a light and hydrophobic organic phase as the stationary phase, and a heavy and hydrophilic aqueous phase as the mobile phase. Hydrophobic interactions between the solvent system and solutes (targets) will increase the partition coefficients (K values) of solutes and lengthen retention time, while hydrophilic interactions will reduce the K values and separation time. In this work, we aim to provide a general insight on the hydrophobic and hydrophilic interactions in CCC separation. We also highlight the current advances in utilizing the hydrophobic and hydrophilic interactions for K-targeted CCC separation and purification. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Liquid chromatography plays a central role in the fields of separation science, such as paper chromatography, thin layer chromatography (TLC), normal phase liquid chromatography (NPLC), reversed phase liquid chromatography (RPLC), ion chromatography. Especially, high-performance and ultra-performance liquid chromatography (HPLC and UPLC) provide more rapid and higher efficient separation, identification and quantification of each component in a mixture. Usually, components in the sample have been eluted out with different rates according to their different degrees of interactions with the adsorbent material in the column of liquid chromatography. These interactions include hydrophobic (dispersive), dipole-dipole, ionic and so on. Among of these interactions, hydrophobic and hydrophilic interactions have been widely applied in the liquid chromatography. Historically, hydrophobic interaction chromatography (HIC), R Selected paper from the 10th International Conference on Countercurrent Chromatography, 1-3 Aug, 2018, Braunschweig, Germany. ∗ Corresponding author. E-mail address: [email protected] (S. Wu).

was first described by Shepard and Tisulius in 1949 using the term “salting out chromatography” [1], and was coined later by Hjerten [2]. HIC separation is usually accomplished by adsorbing the hydrophobic solutes in a mobile phase at high ionic strength (i.e., “salting out”) and desorbing the solutes by reducing the ionic strength, extensively exploited by biopolymer science and ion-exchange chromatography as well as protein separation. In contrast, hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC) suggested by Alpert in 1990 [3] and found to be suitable for adsorbing the hydrophilic compounds, where the analytes interact with the hydrophilic stationary phase and are eluted with a relative hydrophobic binary eluent, with the main components usually being 5–40% water in acetonitrile (ACN) [3]. This is a valuable alternative of reversedphase liquid chromatography to separate small polar, weakly acidic or basic compounds on the polar stationary phase. Besides in solid-support liquid chromatography, recently we noted that hydrophobic and hydrophilic interactions were also widely presented in the separation process by countercurrent chromatography (or counter-current chromatography, CCC). As is well known, CCC is a unique support-free liquid-liquid chromatography and just relies on the partition of solutes between two immiscible

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solvent phases. Thus, CCC provides an advantage over the traditional column chromatography: (i) no irreversible adsorption of the sample; (ii) quantitative recovery of the injected sample; (iii) tailing is minimized; (iv) low risk of sample denaturation; (v) low solvent consumption; (vi) the technique is very economical (after the initial investment in an instrument, no expensive columns are required and only common solvents are used) [4]. In addition, CCC can be easily coupled with other online separation techniques [5]. It should be noted that due to without solid matrix-induced absorption, after each seperation, CCC column is very easy to get fresh and clean only by simply extrusion with fresh two-phase solvents or pure solvents such as water and ethabol. As contrast, for other chromatographic techniques such as NPLC and RPLC, the column is difficult to get back to fresh and clean status even using stong solvent elution, and thus resulted in decreasing resoultion, increasing column pressure and short service life. Up to now, CCC has been widely applied in analysis and separation of various natural and synthetic products, especially some special compounds, such as high polar and unstable compounds. Due to without solid support in CCC column, two-phase partition of solutes in CCC separation were sensitive to the changes of solvents components or additives, which resulted in direct hydrophobic and hydrophilic interactions. So far, several highspeed and high-performance CCC instruments such as helical coils [6,7] and spiral coils or disks assembly [8,9], conical coils [10,11], concentrical coils CCC [12], cross-axis and non-synchronized CCC apparatus [13,14] have been developed during the past forty years, however, the successful CCC separation still largely depends on the selection of two-phase solvent systems. Generally speaking, two key factors should be considered before CCC separation. One is to compose a solvent system with a good two-phase separation, which is the results from the solvent-solvent interaction with/without additives. The used solvent system has clear two liquid phases (upper light phase and lower heavy phase) and short settling time (the seperation time of two phases). Usually, the settling time of two phases was less than 30 s required for a successful type-J CCC separation. The other factor is to make the solutes to have appropriate partition coefficients (K) in the solvent systems, which depends on the whole interaction between two phases and solutes (targets). The suitable K values for highspeed CCC are in the range of 0.5 ≤ K ≤ 1 [15], and the “sweet spot” is centered at K = 1, while 0.4 < K < 2.5 represents a conservative range [16]. In the extrusion mode such as elution-extrusion CCC [17], the “sweet spot” can be significantly extended toward higher K ranges (0.25 < K < 16) [18]. Therefore, the purpose of this work is to provide a general insight on the hydrophobic and hydrophilic interactions in twophase separation and solutes partition for CCC separation. We also highlight the current advances to use the hydrophobic and hydrophilic interactions for targeted purification. To the best of our knowledge, this is the first document to focus on the hydrophobic and hydrophilic interactions in CCC separation. 2. Solvents-induced hydrophobic and hydrophilic interactions As descried above, to form well separated two liquid phases is the first key factor for CCC separation. Since the pioneer invention of CCC by Yoichiro Ito in 1970s [19,20], a large number of two-phase solvent systems have been explored as two immiscible phase for CCC separation. For example, there are several systematic two-phase solvent systems such as Oka’s solvents [21], Abbott’solvent systems [22], HBAW solvent system [23], Ito’s solvent systems [24], ARIZONA solvent systems [25–27], GUESS-mix guided hexane-ethyl acetate-methanol-water (HEMWat) quaternary systems [16,28] and HEMWat 9 × 9 map [29]. The two-phase solvent systems HEMWat might be the most popular solvent systems.

Fig. 1. Solvent induced hydrophobic and hydrophilic interactions make the line change of water and methanol contents in the lower phase of ARIZONA solvent systems. Data were adopted from the refer [25,27].

2.1. Hydrophobic and hydrophilic interactions in two-phase solvent systems In contrast to liquid chromatography using the solid support matrix as a static phase, CCC separation usually only relies on the two liquid phase partition of solutes (targets). Therefore, the components changes of solvent systems will make significant changes of hydrophobic or hydrophilic interactions between solutes and whole solvent systems. It is well known that ARIZONA solvent system (Table 1) was the one of the most used two-phase solvent system which was first proposed by Margraff in 1995 by combining ethyl acetate–water (1:1, system A, polar) with n-heptane– methanol (1:1, system Z, nonpolar) including 23 kinds labeled with capital letters from A to Z, respectively (except E, I and O) [25–27]. Each composition of the AZ system has the same heptane-ethyl acetate and methanol-water volume ratios. The phase density difference and setting time were important for good liquid stationary phase retention in a given CCC column [30]. As shown in Table 1, due to the hydrophilic interaction, the more water content is in the initial system, the larger polarity of the solvent system is. On the contrary, the more ethyl acetate content is, the more tends the nonpolar. In addition, due to these interactions, there is a significant linear trend (Fig. 1) between the initial amount of water (corresponding to the ARIZONA solvent system) and the contents of water and methanol in the lower phase. What’ more, from the A composition (ethyl acetate-water) to Z (heptane-methanol), the polarity of the system was from hydrophilic to hydrophobic, due to decrease of water content and increase of methanol content in the lower phase [25,26]. As a result, the solvent systems could be classified into four parts: hydrophilic, less hydrophilic, less hydrophobic and hydrophobic systems. 2.2. Interaction-induced linear changes between the phase composition of the solvent systems and the log of solute partition coefficients (K) In the separation of CCC, polar solutes have tended to resolve in the lower aqueous phase that contain much water due to the hydrophilic interaction between the solutes and water and have smaller K values. It has been observed and studied that there are some significant linear tendencies between the phase composition of the solvent systems and the log of solute distribution constants [25,31]. For example, in the reversed-phase mode (aqueous lower mobile phase), there is a very significant linear regression of the log K versus water content (%, v/v) of the lower phase of AZ central compositions.

log K = a(%H2 O ) + b

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A-G is the polar region I; H-M is the less polar region II; N-T is the low polar region III; U-Z is the apolar region IV. The settling times are given for freshly prepared heptane-containing liquid systems (±5 s). The data were adopted from the refer [25,27].

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Table 1 The 23 Arizona system compositionsa and related physical propertiesa.

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Fig. 2. Hydrophobic and hydrophilic interactions in the HEMWat 9 × 9 map-based solvent systems [29]. (A) The candidate two-phase solvents. HE, hexane & ethyl acetate; MW, methanol & water. (B) The linear correlations between the content of solvent and the system number in the relative line groups. (C) The linear correlations between the log K values of the solute and the hexane or methanol numbers of the solvent systems on diagonals AA-II. The representative solute includes 2 (Resorcinol, C6 H6 O2 ), 3 (Phenol, C6 H6 O), 5 (Salicylic acid, C7 H6 O3 ), 7 (Apigenin, C15 H12 O5 ), 8 (Cytochalasin D, C30 H37 NO6 ), 9 (Imperatorin, C16 H14 O4 ), 11 (Honokiol, C18 H18 O2 ). (D) Due to the linear change of between log K and phase components, the sweet solvent two-phase solvent system (Sweet spot, K = 1) thus might be calculated from two K values (points).

As listed in the Table 2, except alkaloid caffeine, log K of most compounds of the GUESSmix and water content (%, v/v) of the lower phase of five AZ central compositions from L (heptane/ethyl acetate/methanol/water 2/3/2/3, v/v) to Q (3/2/3/2, v/v) presented a very significant linear regression (r2 , about 0.99) [27]. What’s more, the slopes of the log K versus percent H2 O are positive, so all solute elution volumes will decrease in the reversed-phase mode when the lower phase water content decreases. The values of slopes vary from liquid system compositions [27].

In our previous work about physicochemical properties of the hexane–ethyl acetate–ethanol–water (HEEWat) liquid system, we also got the similar results as shown in the Table 3 [32]. We chose a series of composition with the same hexane–ethyl acetate ratio (apolar solvents) as 8:2 and ethanol–water ratio (polar solvents) changing from 9:1 to 1:9, taking into account that the organic phase of HEEWat system does not contain large amount of water. The natural log values of the partition coefficients of five tanshinones in the extract of rhizome of S. miltiorrhiza Bunge changed

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Table 2 Slope, intercept, and regression coefficient of the log K versus water content (%% v/v) of the aqueous lower phase [27]. Code

Name

Slope

Intercept

r2

K (L)a

K (Q)

C V M Q F U Z N O E

Caffeine Vanillin Coumarin Quercetin Ferulic acid Umbelliferone Salicylic acid Narigenin Carvone Estradiol log K (L) vs log KO/W log K (Q) vs log KO/W

0.0253 0.0312 0.0375 0.0835 0.0453 0.0483 0.0228 0.0793 0.0453 0.0545 0.4102 0.2825

−2.124 −1.751 −1.662 −4.318 −2.798 −2.689 −0.752 −3.740 −1.405 −2.252 −0.621 −1.338

0.852 0.988 0.987 0.999 0.979 0.998 0.952 0.997 0.986 0.991 0.743 0.294

0.16 0.79 2.08 1.23 0.39 0.73 2.83 2.60 9.70 4.22

0.05 0.19 0.37 0.03 0.05 0.08 0.99 0.07 1.19 0.34

a

L, Q mean the Arizona systems L and Q in Table 1.

Table 3 The experimentally measured KC values of tanshinone compounds in the 8:2:E:W hexane–ethyl acetate–ethanol–water (HEEWat) solvent systems [32]. ln(KC ) = A(%W) + B

Compounds

Solvent system (HEEWat, v/v)

Lower phase water percentage (%% v/v)

8:2:1:9 87.7

8:2:2:8 76.0

8:2:3:7 68.7

8:2:4:6 57.7

8:2:5:5 49.8

8:2:6:4 38.1

8:2:7:3 28.6

8:2:8:2 17.5

Slope A

Intercept B

r2

Dihydrotanshinone I Cryptotanshinone Tanshinone I 1,2-Dihydrotanshinquinone Tanshinone IIA

(35) (130) (150) (274) (278)

15.60 50.00 64.00 115.00 124.00

7.60 23.30 26.30 45.70 53.90

2.40 6.75 7.40 13.80 15.90

0.78 2.06 2.80 4.80 9.00

0.41 1.00 1.10 2.00 2.90

0.22 0.50 0.55 0.93 1.42

0.09 0.17 0.23 0.39 0.66

8.79 9.63 9.59 9.68 8.93

−4.14 −3.60 −3.40 −2.88 −2.21

0.983 0.987 0.989 0.988 0.991

linearly with the water content (%W) of the aqueous lower phase. In HEEWat system 8:2:1:9, the K values in the parentheses, were calculated using the regression line obtained from other seven systems and very large, that meaned compounds essentially located in the organic phase of system and not suitable for separation in the CCC experiments. 2.3. Hydrophobic and hydrophilic interactions in HEMWat map-based two-phase solvent systems The hydrophobic and hydrophilic interactions in two-phase solvent system makes it possible that to search a favorable solvent system is not only by taking advantage of above systematical and practical family tables, but also by use of several statistic or mathematic methods based on the linear tendencies between the water content (%, v/v) of the lower phase and log K. As a result, the retention volumes of the analytes can be predicated before separation experiment and the target components can be fixed in advance. For example, J.B. Friesen and G.F.Pauli provided a practical approach for prediction of CCC distribution constants, K values, by thin layer chromatography (TLC) to determine the optimum HEMWat solvent system [33]. And additional information can be acquired from equivalent solvent systems by calibration with the GUESS standard compounds. Recently, we have proposed a HEMWat 9 × 9 map-based strategy to target a suitable solvent system for CCC separation [29], which contains 62 usable two-phase solvent systems as candidate solvent systems (marked in light green in Fig. 2A). There are two significant linear correlations. One is between the content of hexane/ethyl acetate of upper phase or methanol of lower phase of solvent system and the system number in the relative line groups (Fig. 2B). The other is between the log K of the solute versus hexane or methanol content of some solvent systems [10,16,32,34]. Due to the numbers of HEMWat solvent table family are in the significant linear changes with solvent composition in each solvent system line group, thus the log K versus system number of solvent system line groups were kept linear (in the Fig. 2, the number of

hexane or methanol represents the system number), such as diagonals AA-II in the Fig. 2C. As a result, it is very easy to obtain an appropriate solvent system with desired K value (i.e., “sweet point” K = 1) by a simple mathematical calculation (Fig. 2D). Here, it should be noted that all hydrophobic or hydrophilic interactions are both confined to the interactions between targeted solutes and two-phase solvents, not the interaction with each other of solvents. As shown in Fig. 2C, it is possible that adding small amount of hydrophobic solvent hexane yielded a smaller K value, resulting in earlier elution. In this example, this is a fourcomponent two-phase solvent system. As shown in Table 1, on the one hand, with increasing of the content of hexane (hydrophobic solvent), the relative content of ethyl acetate in the upper phase decreased. On the other hand, due to the hydrophobic role of hexane, the methanol distributed in the upper phase may be pushed into lower phase, which made the lower phase more hydrophilic or upper phase more hydrophobic. As a result, the whole twophase solvent system got more hydrophillic, and thus the K values decreased. Therefore, in this case, the hydrophobic solvent (hexane) makes the whole two-phase solvents more hydrophilic and yields smaller K values. In other word, this still lies on the hydrophilic interaction although added hexane is hydrophobic solvent. Interestingly, as shown in Fig. 3A, there are significant linear trends of the components of two phases of the sweet spot solvent systems from different solvent system line groups calculated by pre-set K = 1. Clearly, there are infinite two-phase solvent systems on the trend-line suitable for CCC separation. Thus the result extends the selection range of appropriate solvent system from classical “sweet spot” to “sweet line” (Fig. 3A). Furthermore, A “sweet zone” (Fig. 3B) between the sweet lines i.e., K = 0.5 and K = 3 may be obtained, which contains infinite suitable solvent systems for CCC separation. Therefore, in the model of Fig. 3C, utilizing the linear regression equation through two classical sweet solvent systems SS1 and SS2 , we can calculate any desired solvent system SSx on this sweet line and choose better sweet solvent system with higher sample capacity. Typically, as shown in the

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of solvent systems with appropriate K values of solutes for CCC separation. We can choose systems composing of different solvents from systematical and practical family tables such as HEMWat family [33] and HEMWat 9 × 9 map [29], whose polarity linearly changed. It was fit for separation and purification of a wide range of organic compounds with low and medium polarity. The system of n-hexane–ethyl acetate–n-butanol–methanol–water covers a broad range in both hydrophobicity and polarity continuously from n-hexane–methanol–water to n-butanol–water. Therefore, for compounds with high polarity, n-butanol–water system was usually the best choice. While for hydrophobic compounds, chloroform–methanol–water supplied moderate hydrophobicity and lipophilic systems like hexane-acetonitrile system was found to work best. Some new green two-phase solvents i.e. nhexane/acetonitrile/benzotrifluoride (BTF) [35] provide promising applications. Besides that, we can modify the volume ratio of the solvents in the same biphasic solvent systems family tables relying on the computational K values by HPLC or TLC-based mathematic methods. 3. Additives induced hydrophobic and hydrophilic interactions 3.1. pH regulators

Fig. 3. Solvent-induced hydrophobic and hydrophilic interactions produce infinite suitable two-phase solvent systems for CCC separation. (A) The are infinite sweet solvent systems to form “sweet line” for compounds 5, 6, 7, 8, 10, 11 and 12. (B) Any sweet solvent system can be calculated from the sweet line according to the simplified work model [29]. (C) Besides the sweet line, there are more comprehensive solvent systems within “sweet zone” where K values of targets are suitable for CCC separation, i.e. 0.5 < K < 3. Compounds include 5, Salicylic acid; 6, Andrographolide; 7, Apigenin; 8, Cytochalsin D; 10, Taxol; 11, Honokiol; 12, Magnolol.

Fig. 4, selecting tanshinone IIA (14) as an example, it can be easy to find an optimum two-phase solvent system with the higher sample capacity (HEMWat 4.7:5.3:8.3:1.7 for tanshinone IIA) for successful CCC separation. In general, two-phase solvents make potent hydrophobic or hydrophilic interactions with solutes. We should first consider and utilize these interactions to get well two-phase separation

We have been aware that there is a very low risk of peak skewing in the CCC separation of common compounds without employing a solid support matrix. However, for the ionizable compounds, it is well known that there is significant pH-dependent peak skewing (leading or tailing). There are some compounds extremely sensitive to the pH values of the aqueous phase [36], such as benzoic acid [37] and β -blocker amine [38], whose hydrophobicity depends largely on their ionization state. A small pH change can induce a large change in the relative concentration of the acid-base species of the compound. Taking basic alkaloids as example, at low pH, it existed with the ionization state with increased hydrophilicity and at high pH, its hydrophobicity enhanced. Mostly, the ionizable compounds exist two forms including ionic and non-ionic molecular, thus their partition coefficient (K) usually does not be equal to their distribution ratio [37], making poorly predictive capability of distribution ratio by HPLC or TLC-based methods. This problem was resolved in certain extent, when acid was added to the lower aqueous phase to change the pH and reduce the ionization of organic acids in the sample, such as acetic acid, trifluoroacetic acid (TFA) and hydrochloric acid. In parallel, alkaline reagents were also used to separate alkaloids. Besides that, phosphate buffers (PBS), formic acid, sodium hydroxide, sodium iodide, benzalkonium chloride, potassium perchlorate, ammonium sulfate and O-carboxymethyl chitosan (O–CMC) are also used as modifiers of CCC solvent systems [39,40]. Corresponding, several methods such as pH-gradient [41,42] and pH-zone-refining CCC [43–45] have been developed. pH-zone-refining CCC was first introduced by Yoichiro Ito and coworkers, including a retainer and an eluter. The former, an organic acid or an organic base such as TFA and triethanolamine (TEA), added in the stationary phase and the latter added in the mobile phase at a desired molar ratio to establish pH-zone refining. Sometimes, an inorganic acid such as sulfuric acid can also be a retainer with dodecyl amine (a hydrophobic counter ion) to separate highly polar compounds [46]. In contrast, the eluter should be an inorganic acid such as HCl, or an inorganic base, such as NH3 . H2 O. The formation and traveling rate of the sharp retainer border are two key elements of pH-zone-refining CCC. The latter should be adjusted by the retainer/eluter molar concentration ratio, regardless of the compositions of the biphasic solvent system [44]. In the Fig. 5, both mobile and stationary phases move countercurrent to each other by passing through the TFA (CF3 COOH)

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Fig. 4. High capacity CCC separation using the HEMWat solvent map-based the hydrophobic or hydrophilic interactions [29]. (A) The sweet solvent systems (K = 1) and (B) their linear plot; (C) relative tanshione IIA solubility in these sweet solvent systems (on the top photo, for crude tanshinones); (D) The targeted CCC separation using the optimum solvent system A of HEMWat 4.7:5.3:8.3:1.7 (v/v).

Fig. 5. Portion of the separation column showing analyte movement around a retainer border which is set stationary [47]. The added acid changed the equilibrium of hydrophobic and hydrophilic interactions, yielded a pH-zone-refining CCC separation.

border [47]. On the right side of the TFA border, where the pH is low, all solute S (RCOOH) in the flowing mobile phase is immediately protonated into the hydrophobic nonionic form. It is then quickly transferred into the stationary phase and sent back to the left side of the TFA border. On the left where the pH is initially high, the solute is mostly distributed to the mobile phase. With a continuous supply of the solute from the mobile phase, the solute concentration on the left side of the retainer border starts to rise. The high concentration of solute causes a decrease in pH which in turn increases the partition coefficient of the solute (due to its nonlinear isotherm) until a steady state partition equilibrium is established between the two phases. And in the equilibrium the con-

centration of ionized components in the organic phase is negligible. Consequently, the analyte is always confined in a narrow region around the sharp retainer border and elutes as a sharp peak together with the sharp retainer acid border. pH-zone-refining CCC has been generally applied in preparative separation of ionizable compounds including organic acids and alkaloids based on their pKa values and hydrophobicities, as well as their partition coefficient (KS ) [48]. For example, it was at first used to separate alkaloids from embryo of the seed of Nelumbo nucifera Gaertn (Fig. 6A). The system composed of n-hexane-ethyl acetate-methanol-water (5:5:2:8, v/v), 10 mM trimethylamine in organic stationary phase and 5 mM HCl in aqueous mobile phase,

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Fig. 6. pH-zone-refining countercurrent chromatogram and HPLC control for the separation of the crude extract. (A). Preparative separation of alkaloids from embryo of the seed of Nelumbo nucifera Gaertn [45]. Compound A was liensinine; Compound B was isoliensinine; Compound C was Neferine. (B). Separation of the crude extract of Salvia. miltiorrhiza Bunge [49]. Peak A is salvianolic acid B; Peak B is lithospermic acid; Peak C is rosmarinic acid.

which was the optimum for large-scale CCC isolation. Alkaloids were eluted as an irregular rectangular peak where impurities or minor components were highly concentrated at its front and rear boundaries. The pH measurement of the collected fractions also revealed a flat pH-zone, which correspond to the above absorbance plateaus, suggesting the successful separation [45]. Another successful example was the separation of salvianolic acid B from the Chinese medicinal plant, Salvia miltiorrhiza Bunge, using a multilayer coil planet centrifuge [49]. In the Fig. 6B, a 2.0 g quantity of sample was separated using the following two-phase solvent system: methyl tertbutyl ether (MtBE)-water, 10 mM TFA in organic stationary phase and 10 mM ammonia in aqueous mobile phase. In our previous research, we developed another method that firstly used basic lysine and ammonia as the pH regulator to restrain ionization and keep the polarity of compounds getting an approximate K value. In Fig. 7A and B, the distribution ratios of two phenolic alkaloids obviously increased with the increase of molar concentration of pH regulators, until the phenolic alkaloids existed in the molecular form and the distribution ratio didn’t change any more. In the largely isolation of phenolic alkaloids of N. Nucifera, we found that comparing with the aqueous ammonia, lysine was presented to be more suitable as the pH regulator [50]. As shown in Fig. 7C, the peak 1 and 2 were well resolved at approximate symmetry, and the targets were isolated well on the base-line. Besides that, pH regulators are also useful to increase the K values of polar compounds in the system, such as ion-pair reagents, which are well known reagents in HPLC and CE [51,52]. Nowadays, isolation and purification of betalains by CCC, which are water soluble plant pigments, usually employ solvent systems with ion-pair reagents such as trifluoroacetic acid (TFA) and heptafluorobutyric acid (HFBA). The positively charged betalains created ionpairs with negatively charged counter-ions provided by ion-pair reagents (TFA or HFBA) leading to decrease of their polarity and increase retention. 3.2. Salts induced hydrophobic interactions (salting-out effects) Salting-out is a very common but not simple physical phenomenon extensively exploited by biopolymer science, ionexchange chromatography and HIC, in which, molecules are “forced” out from solution at high ionic strength (i.e., “salting out”). In CCC, “salting-out” or hydrophobic salts reagent such as NaCl,

KCl, (NH4 )2 SO4 , and KNO3 [53,54] was also used to separate polar compounds. Much more than that, these two-phase solvent systems possessed advantages of higher polarity compared with conventional organic-aqueous solvent systems and lower viscosity compared with aqueous polymer systems [55]. The main criterion for salt choice is the salt overall electrostrictive or salting-out capacity. Fig. 8 shows the electrostrictive power of some effective salting-out electrolytes on the distribution constant of 5-CQA in an ethyl acetate-salt (aq, pH 2.5) biphasic system [56]. The relative salting-out power for the different salts is very much in agreement with the Hofmeister series for cations and anions [57]: F− > PO4 3− > SO4 2− > AcO− > Cl− > NO3− > I− > CNS− and Mg2+ > Ca2+ > Ba2+ > Li+ > Na+ > K+ > NH4 + > Cs+ , and directly correlated with the ion hydration enthalpies. As predicted by these series ions with the greatest charge density, e.g. HPO4 2− , SO4 2− and Li+ , proved to be the most electrostrictive, with KNO3 on the lower limit in the set showing a slight salting-in effect. Most likely due to metal complex-derived salting-in effects on the catechol group [58,59] some tightly hydrated and hence supposedly good kosmotropic cations, such as Mg2+ , Ca2+ and Al3+ , were not effective salting-out agents for 5-CQA. Similar phenomena may explain the inverted effect observed for Li+ at extremely high salt concentrations. The distribution constants for all isomers in three most efficient candidate solvent systems adding Lithium chloride for CGAs are summarized in Table 4. Using the salty aqueous layer as a mobile phase in descending mode, the CGA-rich green coffee bean extract was successfully fractionated into the major CGA subfamilies by a LiCl salting-out gradient (Fig. 9A). The stepwise variation included three salt concentrations, 5.0, 2.5 and 0.1 M, applied in decreasing electrostrictive order, thus emulating a conventional reversed-phase gradient. Non electrostrictive salts, KNO3 , was introduced to stabilize the biphasic system as the (NH4 )2 SO4 concentration is reduced, and the settling time was determined for several KNO3 concentrations. The result of this multistep and multi salt method (Fig. 9B) was a separation with a resolution comparable to that obtained with LiCl (Fig. 9A). In our previous research, in the Fig. 10, adding NaCl into the two-phase system with equal volume of ethyl acetate and saltcontaining aqueous solution, it was found that the partition coefficient of arctiin rised with the increase of NaCl concentration, and the K values reached 0.9–1.2 when concentration of

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Fig. 7. pH regulators-induced interactions on phenolic alkaloids of N. nucifera GAERTN in HEMWat system (5: 5: 4: 6, v/v) [50]. (A) ammonia and (B) lysine changed K values the of phenolic alkaloids. Iso, isoliensinine; Nef, neferine. (C) Significant CCC separation could be achieved by adding1 mM lysine, which almost eliminated the peak tailing concentration. Table 4 Hydrophobic interaction (salting-out) effect of lithium chloride on chlorogenic acids in different solvent systems [56]. [LiCl] (M)

3-CQA

5-CQA

4-CQA

CA

5-FQA

4-FQA

3,4-di CQA

3,5-di CQA

4,5-di CQA

Chloroform–n-butanol (82:18) 0.0 14 0.1 0.5 15 2.0 5.0 18 7.0

Settling time (s)

0.01 0.01 0.02 0.03 0.09 0.12

0.12 0.13 0.14 0.30 0.87 1.03

0.06 0.06 0.07 0.15 0.43 0.49

2.92 2.94 3.60 7.19 27.63 41.60

1.77 1.83 2.25 4.57 12.92 13.78

0.74 0.76 0.94 2.01 5.78 6.00

0.54 0.53 0.71 2.35 18.37 24.82

1.53 1.52 2.08 7.03 ≥ ≥

5.49 5.63 7.57 22.83 ≥ ≥

Methyl tert-butyl ether 0.0 10 0.1 0.5 8 2.0 5.0 6 7.0

0.02 0.02 0.03 0.04 0.07 0.06

0.12 0.12 0.18 0.27 0.46 0.35

0.05 0.06 0.09 0.13 0.23 0.16

12.15 13.77 17.41 32.51 ≥ ≥

0.21 0.23 0.34 0.58 1.19 1.02

0.11 0.12 0.18 0.29 0.58 0.47

2.44 2.59 4.18 10.19 36.20 37.12

6.81 7.51 10.76 27.79 ≥ ≥

11.83 13.52 20.24 47.46 ≥ ≥

Ethyl acetate–hexane (70:30) 0.0 17 0.1 0.5 15 2.0 5.0 11 7.0

0.01 0.01 0.01 0.02 0.03 0.03

0.04 0.03 0.05 0.10 0.22 0.16

0.02 0.02 0.03 0.06 0.13 0.09

3.15 3.03 4.55 8.93 24.27 33.04

0.15 0.15 0.22 0.44 1.14 0.91

0.09 0.08 0.13 0.26 0.64 0.50

0.41 0.38 0.67 2.16 12.78 10.57

1.27 1.29 2.14 7.04 ≥ ≥

1.76 1.8 3.09 10.20 ≥ ≥

CA: caffeic acid.

NaCl was up to 5–8% (NaCl:water, w/v), which was suitable for separation of arctiin by the CCC method. And in the system of n-butanol-NaCl aqueous solution, this tendency was also confirmed. Besides that, during the separation of flavonoids in alfalfa, two one-component/salt-containing solvent systems of isopropanol/NaCl and isopropanol/(NH4 )2 SO4 had been investigated.

As shown in Fig. 11A, the volume ratio of isopropanol and the settling time of formed two-phase decreased with the increase of the content of both NaCl and (NH4 )2 SO4 . And the partition coefficients of flavonoids were measured with different systems in Table 5. May be due to hydrophobic interactions (salting-out) in the isopropanol/(NH4 )2 SO4 system, large water distributed into

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Fig. 8. Hydrophobic interactions of salts on chlorogenic acid, 5-CQA, in an ethyl acetate-aqueous salt solution (pH 2.5) biphasic system [56].

Fig. 9. Step-gradient changes of salts-induced hydrophobic interactions made good CPC separation of the major chlorogenic acids present in green coffee beans using (A) LiCl and (B) (NH4 )2 SO4 /KNO3 [56].

Table 5 Hydrophobic interaction of salts on phase seperation in water soluble liquid [61]. No.

Solvent system

Phase ratio (upper:lower, v/v)

Settling time (s)

Partition coefficient (K) of flavonoids DHFGc

MHFG

Ammonium sulfate–isopropanol 1 15%a , 2:1b 1:1 2 15%, 5:5 1.2:0.8 3 35%, 6:4 0.9:1.1

39 15 16

6.15 26.07 46.51

7.63 49.25 89.84

Sodium chloride–isopropanol 4 15%, 5:5 1:01 5 15%, 3:5 1.5:0.5 6 20%, 5:5 1:1

18 10 8

1.09 1.02 1.05

1.24 1.32 1.42

a b c

The concentration of salt aqueous solution (salt:water, w/v). Volume ratio of salt-containing aqueous solution and isopropanol. DHFG, 6.8-dihydroxy-flavone-7-O-β -d-glucuronide; MHFG, 6-methoxy-8-hydroxy-flavone-7-O-β -d-glucuronide.

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CCC separation had been performed in the Fig. 11B and two major flavonoids were well resolved. In order to conveniently remove the salt in a salt-containing solvent system, we established a new hybrid two-dimensional counter-current chromatography and liquid chromatography (2D CCC × LC) system for continuous purification of one target [60] and stop-and-go 2D CCC × LC for multi-component natural products [61]. The first-dimensional CCC column has been designed to fractionalize crude, and the seconddimensional LC column has been packed with macroporous resin for on-line adsorption, desalination. 3.3. Sugar-induced hydrophobic or hydrophilic interactions (sugaring-out or in effects)

Fig. 10. Sodium chloride-induced hydrophobic interactions on arctiin in ethyl acetate-water and n-butanol-water systems. Data were adopted from [60].

the upper phase and following two polar flavonoids were also major distributed. While in the system of isopropanol/NaCl, two flavonoids showed the good partition coefficients (in the range of 0.5–2). Therefore, using the optimized solvent system of isopropanol and 20% NaCl aqueous solution (1:1, v/v), the classical

Small molecular weight sucrose was widely used in the technology named as sucrose gradient centrifugation to purify enveloped viruses and ribosomes as well as to separate cell organelles from crude cellular extracts. And it has many hydrophilic hydroxyl groups (—OH). Therefore, to polar compounds in the biphase system, sugars were added to complete the hydrogen bonds between the analytes with water, resulting that the analytes were pushed into the organic phase, which was similar to salting-out principles and the organic solvents (i.e., ethyl acetate) were also pushed out of the aqueous phase [62], Therefore the role was coined as “sugaring-out” [63], or “sugaring-in” effects. In the previous work (Fig. 12) [63], we found that almost all of

Fig. 11. Hydrophobic interactions of salts on water soluable solvents systems. (A) Adding salts can form well two phases (Phase diagram of isopropanol/system). Isopropanol/NaCl (◦) and isopropanol/(NH4 )2 SO4 (). (B) Two falvonoids can be separated by the salting-out methods using isopropanol-20% NaCl aqueous solution (1:1, v/v) [61]. DHFG, 6,8-dihydroxy-flavone-7-O-β -d-glucuronide; MHFG, 6-methoxy-8-hydroxy-flavone-7-O-β -d-glucuronide. Solvent system.

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Fig. 12. Sugar-induced hydrophobic interactions (Sugaring-out effects) on the components of Dysosma versipellis [63]. (A) Sugaring-out effects on the partition coefficients of the selected compounds in HEMWat systems of 4.5: 5.5: 4.5: 5.5 (v/v). (B) Sugaring-out effects of different types of sugars on the partition coefficients of compound 7 in HEMWat systems of 4.5: 5.5: 4.5: 5.5 (v/v). The representative CCC profiles (C) without sucrose and (D) with 20% sucrose using the HEMWat solvent system (4: 6: 4: 6, v/v). 1, 3,4-Dihydroxybenzoic acid; 2, p-Hydroxybenzoic acid; 3, α -Peltatin; 4, Quercetin; 5, Isopicropodophyllotoxone; 6, β -Peltatin; 7, Podophyllotoxin; 8, Kaempferol; 9, Podophyllotoxone; 10, Podoverine A; 11, Podoverine D; 12, Podoverine E; 13, Podoverine H.

sugars including the monosaccharide and disaccharide could increase the values of the partition coefficients of almost all of targets of the Traditional Chinese Medicine Dysosma versipellis in the two-phase hexane-ethyl acetate-methanol-water solvent systems. What’s more, nine sugars with different chemical structures, including sucrose seemed to have different sugaring-out effects on the resolution and selective separation of some components of D. versipellis. In the classical CCC separation, system with 20% sucrose has been performed and 13 major flavonoids have been well resolved. However, some oligosaccharidest, such as β -cyclodextrin (β CD) and OH-β -CD were found to have different roles in the partition of components of D. versipellis in HEMWat systems of 4.5: 5.5: 4.5: 5.5 (v/v). It is well known that β -cyclodextrin has high stereo selectivity and chiral recognition ability and thus was widely applied in chiral separation by CCC [64,65]. However, in our ex-

periment (Fig 13), we found that, β -CD might have the “sugarout effect” as same as monosaccharide and disaccharide, while its analogue OH-β -CD plays a hydrophilic role (sugaring-in) for the partition of the components. It may be due to that more modification of hydrophilic hydroxyl groups (-OH) results in stronger hydrophilic interactions between solutes (targets) and solvents systems and smaller K values, and decrease of the values of the partition coefficients. Besides above, some high molecular weight polysaccharides and their derivatives, i.e., O-carboxymethyl chitosan (O–CMC), a derivative of macromolecular polysaccharide biopolymer chitosan, have been found to have the same hydrophobic effects and could improve peak resolution of CCC. And K values of solutes like quercetin, luteolin and kaempferol increased with the increase of O–CMC concentration in the stationary phase basically [40]. Tentatively this can be interpreted such that amino and carboxyl groups

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Fig. 13. Different interactions of β -cyclodextrin and its derivative on the partition coefficients of Dysosma versipellis in HEMWat systems of 4.5: 5.5: 4.5: 5.5 (v/v). Data were from our unpublished work. Clearly, β -cyclodextrin showed hydrophobic interactions and made K values bigger as same as common sugar, but the OH-β -cyclodextrin showed hydrophilic interactions and made K smaller.

of O–CMC as active reaction sites, combined other compounds by hydrogen bonds; furthermore, the amino and carboxyl groups can dissociate to form cationic and anionic polyelectrolytes, respectively, which could form electrostatic interaction with negatively or positively charged compounds, respectively. In a word, some sugars like the monosaccharide, disaccharide and oligosaccharide, even derivative of polysaccharide have the potency to be the sugaring-out roles. However, in contrast to the “salting-out” strategy using salts, the “sugaring-out” strategy using sugars is greener and more environmentally friendly, and it does not produce some complications of salty corrosion. Indeed, if a two-phase solvent system is found to have favorable K values (“sweet spot”) after adding sugars, the system may be called a genuine “sweet solvent system”.

3.4. Ionic liquids and organic salts induced hydrophilic interactions (salts induced salting-in effects) Ionic liquids (ILs) have been successfully utilized in many areas of fundamental and applied chemistry for sample preparation before analysis [66–70]. Besides that, ionic liquids may form biphasic liquid systems with numerous solvents, including water, and may play a “salting-in” role, reducing the K values of compounds. There was a special ion effect existing between hydrophobic ionic liquids and water, which displays a direct ion binding with the hydrophobic moiety of the solute stabilizing this species in solution and induces salting-in effect of ionic liquids [71]. In our previous work as shown in the Fig. 14A [72], several ILs ([AMIM]Cl, [MAMIM]Cl and [BMIM]Cl) could increase the solubility of arctiin in the lower phase, and dose-dependent decrease K values of arctiin. We can easily achieve a suitable solvent system with sweet K spots such as K = 1.74 by adding appropriate concentration of ILs instead of adjusting the component proportion of different solvents. In a room temperature IL-based salting-in strategy for CCC separation of arctiin (Fig. 14B), the separation time is greatly shortened when the K value is decreased with the increase of the concentration of the ionic liquids. The separation time can be shortened from 400 min to 150 min. Liu et al. successfully isolated and purified flavonoid compounds by CCC, baicalein-7-O-diglucoside and baicalein-7-Oglucoside, from Oroxylum indicum with ILs as the modifier in the system of ethyl acetate-water-[C4mim][PF6] (5:5:0.2, v/v) [53]. Another successful example was that flavonoid compounds (Baicalin and wogonoside) from Scutellariae Radix were isolated and purified by the same solvent system and IL as modifier.

3.5. Hydrophobic and hydrophilic interactions in the systems with other additives As for the purification of biological macromolecules, the aqueous polymer is used to purified proteins and nucleic acids so on with high polarity [73]. Due to that the polymer has strong hydrophilic property and tends to solubilize in the lower aqueous phase, once polymer is added, compounds (targets) with high polarity have been pushed out from the lower phase into the upper phase, and thus K values are increased, which is also hydrophobic interaction. Among many types of aqueous-aqueous polymer phase systems available, polyethylene glycol (PEG)-inorganic salt system (phosphate or sulfate) and the PEG-dextran (DEX) system are the most commonly used [74]. With PEG-potassium phosphate systems, the cross-axis CPC has been successfully used for the separations of a variety of protein samples, including a mixture of cytochrome C, myoglobin, ovalbumin and hemoglobin [75], human plasma lipoproteins (HDL, LDL and VLDL) [76,77], various recombinant enzymes [78] from E. coli lysate. The advantage of the PEG-phosphate system is that small molecules are mostly partitioned unilaterally either in the upper or lower phase while macromolecules such as proteins are distributed rather evenly between the two phases; hence the small molecule contaminations are largely eliminated from the fraction. As for PEG–inorganic salt systems, the kind of inorganic salt and its concentration can cause the different ionic strength of the systems, thereby affecting the distribution of proteins between the two phases. However, the PEGpotassium phosphate and PEG-ammonium sulfate systems with high salts concentration are not suitable for the separation of proteins, which are easily precipitating out by salts. In this case, other types of aqueous-aqueous polymer two-phase systems composed of PEG (Mr:10 0 0–40 0 0) and dextran (Mr:10,0 0 0 and 40,0 0 0) without salts were needed. A glucosyltransferase (GTF) from Streptococcus mutans (SM) cell-lysate was successfully demonstrated with a 7.5% PEG 3350–10% dextran T40 system containing 10 mM potassium phosphate buffer at pH 9.0 [79]. Besides that, PEG40 0 0phosphate and PEG40 0 0-citrate aqueous polymer two-phase systems with some concentration of sodium chloride had also been used to purify α -amylase from the cultivation supernatant of recombinant Bacillus subtilis by high-speed counter-current chromatography [80]. For organic solutes such as esters, alcohols, aldehydes, ketones, hydrocarbons, and fats, hydrotropes are capable of a severalfold increasing the solubility in the solvent under normal conditions. Adenosine triphosphate (ATP) as a well characterized role in providing energy for biochemical reactions within cells may act as a

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Fig. 14. Ionic liquids showed significant hydrophilic interactions (Salting-in effects) on arctiin of Arctium lappa L [72]. (A) Partition coefficients (K) of arctiin in n-butanolwater (1:1, v/v) decreased with increase of contents of ionic liquids. (B) High concentration of ionic liquids-[AMIM]Cl showed stronger hydrophilic role and resulted in shorter elution using n-butanol-water.

Fig. 15. General model for hydrophobic and hydrophilic interactions in CCC.

new hydrotrope to help solubilize hydrophobic proteins [81]. Hydrotropes can be used to add into the system for the classical CCC separation as the “salting-out” agencies. Other additives like osmolytes, co-solvents and surfactants from small molecules to polymers and from inorganic to organic compounds all have the possible to be the candidates to validly CCC separate, because of hydrophobic and hydrophilic interactions. 4. Conclusions and perspectives In summary, although the terms including hydrophobic interaction CCC and hydrophilic interaction CCC were not been coined in previous studies, in fact hydrophobic and hydrophilic interactions might been found in the common CCC separation. It may be more popular than hydrophobic and hydrophilic interactions in solidsupport liquid chromatography. In each CCC separation, it always needs to select an appropriate two-phase solvent which requires hydrophobic or hydrophilic interaction for solute partition. In order to target an appropriate two-phase solvent system with sweet K values, as shown in Fig. 15, a simple strategy is to add or subtract some agents including solvents, pH regulators, organic or inorganic salts, ionic liquids, sugars, cyclodextrins or other small and largemolecular weight compounds to regulate the interactions between

two solvent phases and solutes. With the concentration increasing of additives, hydrophilic interaction makes K values smaller while hydrophonic interaction pushed the solute to upper phase resulting in increasing K values. Therefore, after limited trial, it is possible to get a favorable solvent system with short settling time and sweet K values. Based on this, in the future, we need to seek for a straight forward approach for selection of CCC solvent systems only referring to many existing selection methods and evaluation systems of solvent systems. So, it is wonderful to screen solvent systems with the help of computer simulations or other means by artificial intelligence. Acknowledgements This work was supported in part by the National Natural Science Foundation of China (grant no. 21672188) and Zhejiang Province (grant no. LY16B020 0 04). Part of this research was presented orally at the 10th international conference on countercurrent chromatography CCC 2018 held at Technische Universität Braunschweig, Braunschweig, Germany, in August 1–3, 2018, which was successfully organized by Prof. Peter Winterhalter and Dr GeroldJerz. We are also indebted to the responses and discussion with colleagues attending this biennial CCC meeting.

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