686 ENANTIOMER SEPARATIONS NMR study confirmed that 2-propanol displaces hexane more efficiently from the polymeric selector, forming more ordered ‘‘solvent-CSP complexes’’ [54]. If the mobile phase is changed, sufficient equilibration time should be allowed to ensure the complete removal of the polar solvent. In order to achieve reproducible results, when switching from an additive-free to an additive-containing mobile phase (e.g., 0.1% trifluoroacetic acid or diethylamine), and vice versa, a prolonged equilibration time may be required [55]. In rare cases the use of particular additives may trigger transient or even persistent conformational changes in the polymeric structure of a polysaccharide derivative, either enhancing or attenuating its initial enantioselectivity. Persistent changes in selectivity were demonstrated to occur with amylose-based CSPs after operation with NP-type mobile phases that include diethylamine [56]; however, flushing with 2-propanol was shown to restore the original selectivity. Acid treatment of Chiralpak AD-H and Chiralcel OD-H CSPs also changed their performance, which was largely restored by washing with amine-containing mobile phases. Changes in enantioselectivity may also occur due to temperature-induced conformational changes of the selectors. Although primarily used in the NP-mode, polysaccharide CSPs have been demonstrated to possess multimodal applicability. Besides the NP-mode and the already mentioned RP-mode [57], these columns can be used in the polar-organic mode (PO mode). The PO mode utilizes nonaqueous mobile phases that are made up of polar organic solvents, such as methanol or acetonitrile or a mixture of both, to which small amounts of organic acids (acetic acid, formic acid) and base (triethylamine, diethylamine, ammonia) are added as buffer constituents or competing agents. An important benefit of the PO mode, as opposed to normal-phase separation, is a better compatibility with (increasingly popular) electrospray MS detection, as well as better solubility of the analytes in the polar-organic mobile phase, and polar-organic sample matrix. A change in enantioselectivity is often observed for the same compound upon a switch from normal-phase to reversed-phase mode [47]. As already noted, dedicated versions of Chiralpak AD-RH, Chiralcel OD-RH, and Chiralcel OJ-RH have been introduced for reversed-phase applications [57], coated onto a support that is compatible with RP separations. For neutral analytes, simple water-organic mixtures can be used (with acetonitrile being preferred to ethanol, 2-propanol, or methanol). While basic or acidic additives have little effect on the separation of neutral solutes, ionizable compounds, in contrast, require such additives to improve peak shapes. Phosphoric acid (pH-2) is recommended for acidic analytes. Enantiomer separation of basic analytes may be enhanced by addition of chaotropic counter-ions, such as perchlorate or hexafluorophosphate. Alternatively, basic buffer systems at pH-9 (preferably the relatively mild borate) can be employed to efficiently suppress dissociation of basic analytes [57]. For applications requiring mass-sensitive detection, volatile buffer systems must be used. For acidic analytes, phosphate can be replaced by formic acid, pH > 3; for basic analytes, borate can be replaced by ammonium bicarbonate buffer, pH-9.0. Nevertheless, it should be kept in mind that extended exposure to pH-9 will inevitably shorten column lifetime, as it is the case with most silica-based stationary phases. Polysaccharide CSPs are also fully compatible with polar-organic mobile phases [58, 59], as mentioned above. A rather comprehensive study reported by Lynam [59] 14.6 CHIRAL STATIONARY PHASES AND THEIR CHARACTERISTICS 687 Standard mobile phases Normal phase conditions: Alkane/alcohol Polar organic mode: Acetonitrile Alcohols (EtOH, MeOH) Nonstandard mobile phases MTBE, Toluene, Choroform, Dichloromethane, Ethylacetate, THF, 1,4-dioxane, Acetone, DMSO (solvent strength increases in this order ) Coated CSPs Immobilized CSPs Forbidden! They will irreversibly destroy the coated CSP Figure 14.11 Allowable mobile phases for polysaccharide-based CSPs. A distinction is made between standard solvents to be used with all CSPs, and nonstandard solvents to be used only with immobilized CSPs (MTBE, methyl-t-butyl ether). Adapted from [60]. demonstrated that out of 80 test compounds, about 30% could be resolved with pure methanol, ethanol, or acetonitrile as mobile phases. Upon addition of hexane, analyte retention was increased as expected, while there was only a modest effect on enantioselectivity. The authors concluded that the chiral recognition processes operating with polar-organic mobile phases may be similar to those for normal-phase conditions. The most critical limitation of the coated polysaccharide-type CSPs is their incompatibility with certain types of solvents, so-called nonstandard solvents, which include (among others) dichloromethane, chloroform, ethyl acetate, tetrahydrofuran, dioxane, toluene, and acetone (Fig. 14.11). The exposure of coated CSPs to the latter solvents induces swelling and/or dissolution of the physically adsorbed polymer layer, with destruction of the column. Such solvents must be strictly avoided, even as constituents in the sample matrix. To overcome this drawback, several research groups have developed fully solvent-resistant, immobilized polysaccharide-based CSPs by means of various procedures [61–63]. Since 2005 a set of three immobilized polysaccharide CSPs have become commercially available from Daicel and Chiral Technologies as: •Chiralpak ® IA (immobilized version of Chiralpak AD) [64] •Chiralpak ® IB (immobilized Chiralcel OD) [60] •Chiralpak ® IC [65] based on the cellulose tris(3,5-dichlorophenylcarbamate) selector that is not available in coated form (see Fig. 14.10) These CSPs have been demonstrated to be fully compatible with any organic solvents, expanding the applicability to nonstandard solvents that cannot be used with the coated CSPs (Fig. 14.11). Moreover this new generation of immobilized polysaccharide-type CSPs also performs very well in PO, RP, and SFC mobile phases. 688 ENANTIOMER SEPARATIONS While the availability of additional solvents provides unique opportunities and considerable flexibility for method development, it necessarily involves more extended and elaborate screening—with additional labor. To address this latter problem, systematic studies have created an efficient generic screening strategy based on the three commercially available, immobilized CSPs and a limited set of mobile phases. Using Chiralpak IA, IB, and IC and only five starting mobile phases for method development (alkane-2-propanol 80:20, alkane-ethanol 80:20, methyl tert-butylether-ethanol 98:2, alkane-tetrahydrofuran 70:30, and alkane-dichloromethane-ethanol 50:50:2), about 90% of a series of 70 randomly selected chiral analytes could be baseline separated [66]. Interestingly this screening strategy with nonstandard solvents and immobilized polysaccharide-type CSPs pro- vided novel enantioselectivity that was not achievable with coated-version columns and standard-type mobile-phase conditions; for specific cases, greatly improved separations resulted [60]. Fully solvent-compatible, immobilized CSPs also offer unique advantages for preparative applications. Specifically, the poor solubility of chiral compounds in standard NP mobile phases is often the most serious bottleneck in the development of preparative enantiomer separation with coated polysaccharide-type CSPs—which, in general, have among the highest loading capacities of all commercially available CSPs [61]. This is particularly true for polar analytes, which are often sparingly soluble in alkane-based NP mobile phases. With immobilized CSPs, strong solvents such as tetrahydrofuran, dichloromethane, chloroform, and even dimethyl sulfoxide can be used for sample dissolution, without any concern for column stability. Residual, nonstandard solvents in the sample are also not an issue. Another beneficial feature of immobilized polysaccharide-type CSPs is that they can be regenerated with strong, nonstandard solvents such as tetrahydrofuran, dimethyl formamide, and even dimethyl sulfoxide, allowing the effective removal of strongly adsorbed sample components, and improving the reproducibility of subsequent separations. A problem can arise if a method that was developed for a coated polysac- charide CSP is transferred to the corresponding immobilized CSP. The proprietary immobilization process appears to modify the enantiomer separation characteristics as compared to the coated versions [60, 67, 68]. For example, enantioselectivity may be lost upon exchange of the (coated) Chiralcel OD column by the (immo- bilized) Chiralpak IB column when using the standard alkane-based mobile phase (Fig. 14.12b vs. 14.12c), although both CSPs are based on the same polysaccha- ride derivative. Nevertheless, after re-optimization of the mobile-phase composition (including use of the tolerated nonstandard solvents), a greatly improved separation can be obtained (compare Fig. 14.12a–b or c). Comparable results were obtained with acidic (Figs. 14.12a–c), basic (Figs. 14.12d–f ), and neutral compounds. These examples demonstrate a certain level of complementarity between coated and immo- bilized polysaccharide-type CSPs, which represents an advantage for both analytical and preparative method development. The combined use of both types of CSPs can provide chromatographers with an even more powerful tool for separation challenges of ever-increasing complexity. Certainly the introduction of the immo- bilized polysaccharide CSP technology represented an important milestone in the development of enantiomer separation. 14.6 CHIRAL STATIONARY PHASES AND THEIR CHARACTERISTICS 689 0 2 4 6 8 10 min 0 2 4 6 8 10 min0246810min 0 2 4 6 8 10 12 min 0 2 4 6 8 10 12 14 min 0 2 4 6 8 10 12 min (a)(b)(c) (d )(e)(f ) Chiralpak ® IB (immobilized) Chiralpak ® IB (immobilized) Chiralcel ® OD-H (coated) hexane/CHCl 3 /EtOH/TFA 68/30/2/0.1 v/v hexane/EtOH/TFA 90/10/0.1 v/v MtBE/EtOH/EDA 95/5/0.1 v/v hexane/EtOH/EDA 70/30/0.1 v/v Figure 14.12 Effect of column type (immobilized vs. coated polysaccharide-based CSP) and mobile phase on enantioselectivity. Enantiomer separations of N-benzyloxycarbonyl-phenylalanine (a–c) and laudanosine (d–f ) with Chiralpak IB (immobilized) and Chiralcel OD (coated). Flow rate, 1 mL/min; temperature, 25 ◦ C; UV detec- tion at 230 nm. Note that the mobile phases used in (a)and(d) are forbidden mobile phases for the coated version Chiralcel OD. (EDA, ethylenediamine; MtBE, methyl tert-butylether). Reprinted with permission from ref. [60]. Finally, it should be noted that polysaccharide CSPs have now also been established as the first-choice of chiral phases for SFC enantiomer separation [69–72]. 14.6.2 Synthetic-Polymer CSPs A number of chiral synthetic polymers have been proposed as potential selectors, in an attempt to mimic the enantioselectivity of the semi-synthetic polysaccharides of Section 14.6.1. Like the polysaccharides, chiral synthetic polymers are constructed from identical chiral building blocks. However, the synthetic polymers do not achieve the same enantiorecognition as found for the polysaccharide CSPs. This may be the result of a much less ordered structure of the polymer chains, as well as the lack of pre-organized grooves and clefts for solute insertion. Evidence that an ordered chiral hyperstructure is alone sufficient for effective chiral recognition has been furnished by Okamoto et al. [73]. A CSP was prepared from single-handed helical poly(triphenylmethacrylate), obtained by anionic poly- merization from essentially achiral monomers (using a chiral catalyst); the polymer 690 ENANTIOMER SEPARATIONS N N isotactic polymerization (−)-sparteine or other chiral catalyst (b) (a) ChiraSpher (Merck, Darmstadt, Germany) Chiralpak OT(+) or OT(−) (Daicel/Chiral Technologies) C OO OO CH 3 H 2 CC CH 3 n 01051520 ( min ) N N H O O CI O S CH 3 Figure 14.13 CSPs based on polymethacrylate-type chiral polymers. (a) Helically chiral poly(tritylmethacrylate) based CSP obtained by isotactic anionic polymerization in the pres- ence of sparteine as catalyst (Chiralpak ® OT(+)orOT(−) from Daicel and Chiral Technolo- gies,). (b) poly[N-acryloyl-(S)-phenylalanine-ethyl ester] (ChiraSpher ® from Merck KGaA, Darmstadt, Germany). Chromatographic conditions in (b): Column dimension, 250 × 4.6 mm column; mobile phase, 80:20 n-hexane/2-propanol; flow rate, 1 mL/min; temperature, 25 ◦ C. Adapted from [77] (a) and [78] (b). chains were then coated onto macroporous silica (later commercialized by Daicel and Chiral Technologies under trade name Chiralpak OT+; Fig. 14.13a). The latter column successfully separated a number of chiral compounds that possessed multiple aromatic functionalities such as 1,1 -binaphthyl-2,2 -diol (α = 2.0 with methanol as mobile phase at 5 ◦ C). Since this CSP suffers from rather poor chemical stability, it is now more of academic than practical interest. In 1974 Blaschke introduced new polymeric materials in the shape of self-supporting cross-linked poly(meth)acrylamide polymer beads [74], prepared by suspension polymerization of acryl- or methacrylamides, with the chiral element residing as a stereogenic center in the amide side chain. To alleviate exten- sive swelling, and insufficient mechanical stability in high-pressure separations, silica-supported composite materials with more favorable chromatographic per- formances were later developed [75]. The CSP obtained by copolymerization of N-acryloyl-(S)-phenylalanine ethyl ester as monomer with vinylized-silica particles has been commercialized by Merck KGaA (Darmstadt, Germany) under the trade name ChiraSpher (Fig. 14.13b). This CSP, which is still used and has preparative capabilities [61], has been useful for the separation of a variety of polar pharma- ceuticals with hydrogen donor-acceptor functionalities, employing normal phase conditions (usually n-hexane or n-heptane with a polar solvent such as alcohols, dioxane, or THF). A comprehensive review on this topic has been published by Kinkel [76]. 14.6 CHIRAL STATIONARY PHASES AND THEIR CHARACTERISTICS 691 Synthetic polymeric phases are commonly prepared by the classical ‘‘grafting- to’’ approach, that is, formation of the polymer from monomers in solution, with its subsequent anchoring to a support that has been modified by vinyl groups for copolymerization. Frequent problems are encountered with such polymer-type CSPs because of pore blockage and the inhomogeneous distribution of the polymer on the silica surface—leading to lower plate numbers for polymeric CSPs, compared to brush-type phases (Section 14.6.7). Gasparrini and coworkers recently proposed an alternative procedure for the generation of silica-supported polymer-type composite CSPs. Instead of the ‘‘grafting-to approach (Fig. 14.14a), a ‘‘grafting-from technique (Fig 14.14b)was used to attach chiral selector moieties onto the surface of the silica particles [79]. In a first step, the radical initiator (‘‘G’’) is immobilized onto the surface. After the addition of monomers and initiating polymerization by heating, the polymer chains begin to grow from the surface in a more regular way, resulting in a well-ordered surface-confined polymer layer. Detrimental polymer chains grown in solution can be removed during the washing steps. The procedure has been implemented suc- cessfully with (trans-1,2-diamino-1,2-diphenylethane)-N, N-diacrylamide as well as (trans-1,2-diaminocyclohexane)-N, N-diacrylamide as monomers (commercialized as P-CAP-DP and P-CAP in both enantiomeric forms by ASTEC). The synthesis of the latter is shown in Figure 14.14c. CSPs as in (Fig. 14.14b) exhibited a significantly higher column efficiency in terms of the control materials obtained by conventional grafting (i.e., a copolymerization or grafting-to procedure as in Fig. 14.14a). A broad range of chiral compounds, such as benzodiazepines, carboxylic acids and sulfoxides, esters, amides, lactones, and N-blocked amino acids can be separated primarily in the NP or PO mode. A new class of CSPs based on network-type polymers has been proposed by Allenmark et al. [80, 81]. The preparation of these CSPs starts with O, O -diaroyl- N, N -diallyl-(R, R)-tartardiamide as chiral monomers, which are immobilized onto vinylized silica by cross-linking the monomers with multifunctional hydrosilanes— yielding a network polymer that has incorporated the bifunctional C 2 -symmetric chiral selector as a thin film on the silica surface (Fig. 14.15a). Through network formation and multiple crosslinks of the polymer network to the vinylized silica, highly stable CSPs with low column bleed can be obtained (a general benefit of such CSPs). Different substitution patterns of the hydroxy groups can give rise to unique enantioselectivity. CSPs in which the tartramides are substituted with O, O -bis(3,5-dimethylbenzoyl) and O, O -bis[4-(tert-butyl)benzoyl] moieties are commercially available from Eka Chemicals (Bohus, Sweden) under the trade names Kromasil CHI-DMB and CHI-TBB. These CSPs exhibit useful enantiorecognition properties under NP conditions for a variety of pharmaceutically relevant entities, including acidic, neutral, and basic compounds that carry hydrogen donor-acceptor groups and π–π-interaction sites (as in the separation of Fig. 14.15b). 14.6.3 Protein Phases The stereoselective binding of chiral compounds to proteins was an early discovery in biochemistry. Since nature offers a wide choice of stereoselective proteins, their exploitation as chiral selectors for enantioselective liquid chromatography seems obvious. Allenmark, Hermansson, Miwa, Haginaka, and others, have pioneered 692 ENANTIOMER SEPARATIONS SILICA SURFACE CN CN CN CN CN NC N NNC COOH N N CONH CONH NHCO NHCO CONH CONH CONH NHCO SILICA SURFACE SILICA SURFACE silica silica Grafting-to (a) (b) (c) Grafting-from initiator chiral monomer G G G G NH 2 NH 2 NH 2 G G G G (1) (2) Figure 14.14 Preparation of synthetic-polymer CSPs (polyacrylate-type). (a) Grafting-to and (b) grafting-from approach; (c) reaction scheme for the synthesis of poly(diaminocyclohexane- N,N-diacrylamide)-based CSP prepared by the grafting-from concept (b). Adapted from [79]. 14.6 CHIRAL STATIONARY PHASES AND THEIR CHARACTERISTICS 693 (a) (b) COOH S O H R N O O O N H O O R Chiral monomer O HSI SIH SI SI SI SI SI HSI SIH SIH SI SI 0 5 10 min (R) (S) Figure 14.15 Synthetic tartardiamide-derived network-polymer based CSPs. (a) Preparation; (b) separation with 80:20 hexane/THF plus 0.05% TFA. Adapted from [80]. this field. Besides a wide variety of protein-based CSPs that are well documented in the literature [82–84], a number of such protein phases have become commer- cially available. Table 14.2 summarizes the most important protein phases, along with some of their characteristics and column trade names. Among them, α 1 -acid glycoprotein (AGP) [85] and crude ovomucoid (OVM) [86] (commercially available as Chiral AGP and Ultron ES-OVM, respectively) exhibit the broadest enantiomer 694 ENANTIOMER SEPARATIONS Table 14.2 Important Protein-Type CSPs and Column Trade Names, with Some Characteristics of the Protein Selectors Protein Molecular CarbohydrateIsoelectric Column Trade Weight (kDa) (%) Point Name (Supplier) Serum albumin Human (HSA) 67 0 4.7 Chiral HSA (ChromTech) Bovine (BSA) 68 0 4.7 Resolvosil BSA (Macherey Nagel) α 1 -Acid glycoprotein (AGP) 44 45 2.7 Chiral AGP (ChromTech) Ovomucoid (OVM) 28 17-34 4.5 Ultron ES-OVM (Shinwa Chemical) Cellobiohydrolase I (CBH) 60–70 6 3.6 Chiral CBH (ChromTech) Avidin 66 20.5 9.5–10 Bioptic AV-1 (GL Sciences) Pepsin 70–78 - 6.1–6.6 Ultron ES-Pepsin (Shinwa Chemical) Source: Adapted from [83]. separation capabilities, covering a wide variety of neutral, acidic, and basic drugs. The range of applicable analytes is much narrower for other protein-type CSPs, and their use is correspondingly more limited. Cellobiohydrolase I (CBH I) [87] (Chiral CBH) is preferred for basic analytes (e.g. β-blockers), and human serum albumin (HSA) [88] (commercially available under trade name Chiral HSA) is used with acidic analytes. Apart from their use in assaying chiral compounds, HSA-based CSPs have received considerable attention for the study of drug–protein binding because the binding characteristics observed under physiological conditions in plasma are largely maintained when the protein is immobilized onto a silica support. This, unfortunately, does not apply for commercial AGP columns, which display consid- erably altered binding characteristics after immobilization that is due to the specific cross-linking procedure used by the manufacturer to improve column stability. Structurally AGP consists of a single 181-residue peptide chain carrying five heteropolysaccharide moieties. The latter incorporate 14 sialic acid units that render AGP strongly acidic (pI = 2.7) [89]. AGP, covalently attached with cross-linking onto a chemically modified silica surface (by Hermansson [85]), tolerates a wide range of pH changes (3–7.5), high concentrations of organic solvents (up to 25%) and temperature up to 70 ◦ C. Crude chicken OVM-based CSPs, first described by Miwa et al., were later found to owe their chiral recognition ability to an impurity (ovoglycoprotein; 11%; w/w) in the crude OVM [90, 91]. In contrast, pure chicken ovomucoid turned out to have negligible enantiodiscrimination capabilities [92]. OVM-based CSP is stable in the pH range of 3 to 7.5 and tolerates organic solvents up to 50%; the separation temperature should be <40 ◦ C. Protein phases are always used with aqueous or hydro-organic mobile phases (i.e., RP-mode). Mobile-phase pH, buffer type and strength, type and content of 14.6 CHIRAL STATIONARY PHASES AND THEIR CHARACTERISTICS 695 organic solvents, and additives—along with temperature—are the key variables for controlling retention and enantioselectivity. The appropriate conditions must be selected empirically, and depend on solute structure. For acidic and basic drugs, reten- tion will vary with pH as a function of solute and selector ionization; ionic-strength variation can be a powerful tool to vary retention and improve selectivity. An increase in ionic strength can either decrease retention by shielding charges on the analyte or CSP or increase retention by salting out the analyte, thereby enforcing hydrophobic solute-sorbent interactions. The retention of neutral molecules can also be altered by a change in pH, as a result of changes in the protein struc- ture/conformation (leading to altered binding properties). It is known, for example, that OVM can undergo reversible unfolding and refolding as a function of pH [93]. The addition of organic solvents (especially 1- or 2-propanol, or acetonitrile) reduces retention by weakening hydrophobic interactions. Enantioselectivity may thereby either decrease or increase, depending on whether these hydrophobic inter- actions occur at enantioselective or non-enantioselective sites. As an example of the potential effect of a change in organic solvent, verapamil was not separated on the AGP-type CSP with a mobile phase containing 1-propanol but was completely resolved when 1-propanol was replaced by acetonitrile [94]. It should be kept in mind that the addition of organic solvents may cause reversible changes of the sec- ondary structure of the immobilized protein, thereby altering its enantioselectivity. Organic solvent concentrations that are too high can cause irreversible changes and denaturation of the protein, and must be avoided in order not to damage the column. As with any column, the manufacturers’ instructions should be followed strictly. If on-line mixing is used, we strongly suggest ensuring that none of the reservoirs contains organic solvent at a higher concentration than that tolerated by the column, so as to avoid inadvertent destruction of the (expensive!) column Cationic additives (alkylamines or quaternary-ammonium salts, such as N, N- dioctylamine and tetrabutylammonium bromide) and anionic additives (hydropho- bic carboxylic acids, alkylsulfonates), which can compete with solutes for both ionic and hydrophobic interaction sites, have also been used to control retention and enantioselectivity. The addition of 50 μmol/L disodium 1,2-ethylenediamine- N, N, N ,N -tetraacetic acid (EDTA) to the mobile phase is sometimes suggested for the purpose of shielding the protein phase from contamination by metals that may originate from the equipment. Temperature is another variable for the fine tuning of retention and enantioselectivity. Unlike many other columns, van’t Hoff plots of protein phases frequently deviate from linearity (failure of Eq. 14.4). Such unusual temperature behavior can also be attributed to conformational changes of the protein. For example, the temperature dependence of enantioseparations of quinoline-substituted carboxylic acids on AGP suggests such a change in protein conformation between 30 and 35 ◦ C [95]. Because of the structural complexity of these macromolecular protein-selectors and their frequent conformational changes with conditions, not much is known about the fundamental retention process or how this relates to enantioselectivity. Beginning in the early 1980s, protein-based CSPs were popular—especially because they could be used with RP conditions and with aqueous samples, which was favor- able for bioanalytical applications. Due to some major limitations, however, their use has since declined. Significant restrictions exist in terms of mobile-phase compo- sition, as noted above. Improper use or storage, or elongated operation with more . Instead of the ‘‘grafting -to approach (Fig. 14.14a), a ‘‘grafting-from technique (Fig 14.14b)was used to attach chiral selector moieties onto the surface of the silica particles [79]. In a first. ) Chiralpak ® IB (immobilized) Chiralpak ® IB (immobilized) Chiralcel ® OD-H (coated) hexane/CHCl 3 /EtOH/TFA 68/30/2/0.1 v/v hexane/EtOH/TFA 90/10/0.1 v/v MtBE/EtOH/EDA 95/5/0.1 v/v hexane/EtOH/EDA 70/30/0.1 v/v Figure 14.12 Effect of column. conditions: Alkane/alcohol Polar organic mode: Acetonitrile Alcohols (EtOH, MeOH) Nonstandard mobile phases MTBE, Toluene, Choroform, Dichloromethane, Ethylacetate, THF, 1,4-dioxane, Acetone, DMSO (solvent strength