配体交换毛细管电泳-外文文献翻译.docx

英文文献Ligand-Exchange Capillary ElectrophoresisAbstract: The fundamentals of ligand-exchange capillary electrophoresis are discussed, and the potential of the method in solving major problems, such as the separation of enantiomers and the determination of biologically active compounds poorly absorbing in the UV region (sugars, amines, and amino acids) is considered.Keywords: ligand exchange, capillary electrophoresis, complexation, biologically active substances.The separation methods based on ligand exchange are sensitive to the spatial structure of the analyte molecules, which enables the separation of geometric and optical isomers. The method of ligand-exchange chromatography was used in 1968 by Davankov and Rogozhin for separating amino acid enantiomers 1. Polystyrene with immobilized L-proline was used as a stationary phase, while the ions of copper(II) and other transition metals were added into the eluent as complexants. Later, silica-based adsorbents with immobilized optically active ligands came into use 2.One of the promising electrophoretic versions for analyzing complex mixture of natural compounds is ligand-exchange capillary electrophoresis (LECE) 3-19, whose closest analogue is ligand-exchange chromatography 1. Ligand-exchange capillary electrophoresis is mainly used for separating optical isomers of amino and hydroxy acids; in fact, this is a chiral version of LECE 3-21. Chiral separation in LECE, as well as in ligand-exchange chromatography, is based on the formation of diastereomeric complexes with a metal cation between the ligand of a chiral selector (L-Sel) and the analyte enantiomers (L-A and D-A) 5:The formed complexes of analyteMn+ are different in stability, which causes the difference in the electrophoretic mobilities of the enantiomers :where and are the molar fractions and and are the electrophoretic mobilities of the unbound analyte (f) and the corresponding complex (c).Factors affecting the analyte separation.Weak intermolecular interactions causing the formation of labile coordination compounds differing in the electrophoretic and chromatographic characteristics can serve as a basis for separating analytes similar in chemical structure, including enantiomers. For polar compounds, the main reason for forming molecular complexes is specific interactions, first of all, donor-acceptor interactions and hydrogen bonds. Hydrophobic compound can also form molecular complexes, for example, the inclusion complexes of steroids with -cyclodextrin. These processes are due to dispersion interactions. Hydrogen bonds, as a rule, are characterized by a larger interaction energy (10-40 kJ/mol) with respect to the van der Waals forces (10-18 kJ/mol); however, they are much weaker than covalent and ionic bonds (500 kJ/mol) 22.The complex formation is also affected by the ratio of complexant and analyte, spatial complementarity, the effect of medium, probable competing processes, and others.Nature of the coordination center.One of the factors affecting the stability of formed complexes and, therefore, the selectivity of separation and the sensitivity of determination in the methods of ligand-exchange chromatography and ligand-exchange capillary electrophoresis is the nature of both the coordination center and the heteroatoms of interacting ligand. The stability of the complex can be predicted on the basis of the Pearson concept of hard and soft acids and bases 23: the most stable complexes are formed in the interaction of acids and bases of the same degree of hardness, that is, when a hard acid interacts with a hard base and vice versa. Here, softness means the tendency to form complexes with preferably covalent bonds, while hardness, with ionic bonds (Table 1). Therefore, the nature of the analyte determines the selection of the complexing metal.Table 1. Lewis classification of acids and bases 24CompoundsHardIntermediateSoftLewis acids(complexing metals)Li（）, Mg（）, Ca（）, Al（）, Sc（）, rare-earth elementsFe（）, Co（）, Ni（）, Cu（）, Zn（）Cu（）, Ag（）, Hg（）, Pb（）Lewis bases(donor ligand atoms)O, FN, Cl, BrS, P（）, IIn ligand-exchange chromatography, metal ions that are soft acids are the most often used. The Irving-Williams series sets that the stability of complexes increases in the order of Mn < Fe < Co < Ni < Cu for all soft bases serving as ligands. This is the reason why the copper(II) salts are most often added to the eluent or working electrolyte in the determination of amines and amino acids in ligand-exchange chromatography and LECE 1-18. It is also known that polyatomic alcohols (glycerol, sugars, cyclodextrins, and others) can form stable complexes with the Cu2+ cations, in spite of the hardness of the oxygen atoms is considerably higher than that of the copper(II) ions (Table 1).The factor ensuring the stability of such complexes is the chelate effect. The main thermodynamic parameter affecting the increase in stability in chelate formation is the change in the entropy of the system. For example, in the formation of the complexes of cadmium with monodentate (NH3, NH2CH3) and bidentate ligands (NH2CH2CH2NH2), the difference in the values of H° are insignificant, while the values of S° differ considerably (Table 2), which makes a decisive contribution to the stabilization of the chelate complex 24. The greatest advantage of the entropy, according to the Chugaev rule, is observed for the five to six-membered cycles, while for the cycles with the number of atoms more than seven, the advantage is so large, that these cycles are barely formed.Table 2. Effect of chelate formation on the thermodynamic characteristics of the complexes of cadmium(II) with amines at 25°C 24ComplexH°, kJ mol-1S°, kJ mol-1 KG, kJ mol-1°-53.14-35.50-42.51-57.32-66.94-37.41-56.48-13.75-60.67Structure peculiarities of analyte.The structure peculiarities of analyte play a decisive role in the formation of the inclusion complexes, where the spatial complementarity of the analyte molecules and the macrocycle is necessary. The requirements on the structure of the amine component, whose enantiomers should be electrophoretically separated by the interaction with (+)- (18-crown-6)- 2,3,11,12 -tetracarboxylic acid, are described 6, namely, the presence of a primary amino group, the presence of bulky substituents at the asymmetric carbon atom (the presence of several bulky radicals considerably decreases the stability of the complex of amine with crown ether), and the minimal distance between the amino group and the asymmetric center of the amine.The effect of the spatial structure of the analyte is the most pronounced in the electrophoretic and chromatographic separation of enantiomers. It was shown in the first work on LECE, devoted to the separation of amino acid enantiomers as mixed-ligand complexes with the copper(II) ions and L-histidine, that when the latter is substituted by D-isomer, the migration order of the analytes was inversed 3.Composition of the working electrolyte.In the optimization of the parameters of the chromatographic and electrophoretic separation of enantiomers, the pH value of the working electrolyte is an important factor, the variation of which allows the control over the degree of dissociation of both the chiral selector and the analyte according to the values of their isoelectric points (pI). The best resolution of the peaks of enantiomers of amino acids, peptides, and hydroxy acids in their separation by LECE is attained, as a rule, in a weakly acidic medium (pH 4-7) 4, 8-11, 14-18. For example, the optimal value of the resolution factor (Rs) of the enantiomers of histidine (pI = 7.59) is observed at pH 6, while in the separation of the optical isomers of phenylalanine (pI = 5.48), the working electrolyte with pH 4.3 should be used.The effect of pH on the complexation processes becomes more pronounced, when a chiral selector is used bearing several functional groups that can be centers of complexation. It was found that the separation mechanism involving the system of Cu(II)L-prolinamide is more complex than for Cu(II)L-proline. In the latter case, only two different complexes can occur (CuL2+ and CuL22+, Fig. 1), while in the former case, five complexes (CuL2+, CuL22+, CuLH1+, CuL2H1+, and CuL2H2) 17, whose ratio considerably depends on the medium pH, since CuL2+ and CuL22+ occur in an acidic medium (pH < 7), CuLH1+ , at pH 7, and CuL2H1+ and CuL2H2, in an alkaline medium (pH > 7).Fig. 1. Structure of the complexes of Cu(II) with prolinamide 17It was shown that the best resolution of the dansyl amino acid derivatives was observed at pH 6.0 17. This means that complexes CuL2+ and CuL22+ exhibit the highest enantioselectivity. However, low pH values lead to a decrease in the electroosmotic flow rate and, consecutively, to an increase in the time of electrophoretic separation, which should also be considered in the selection of the optimal pH value of the working electrolyte.Concentration and composition of the chiral center.In the selection of the conditions for the electrophoretic and chromatographic separation of enantiomers by the mechanism of ligand exchange, the concentration of the complex and its composition, that is the molar ratio of the complexing metal and the ligand, are important factors. The effect of these factors was thoroughly studied in 17 by the example of the dansyl derivatives of D,L-serine and D,L-valine. It was shown that the resolution of D- and L-isomers was improved with the increase in the concentration ratio of L-prolinamide to Cu(II) from 1 : 2 to 3 : 1. The further increase in the concentration ratio had no significant effect on Rs. Therefore, complex Cu(II)(L-prolinamide)22+ can be considered to be the best selector for the separation of these enantiomers using the principle of ligand exchange. The increase in the Rs value with the change in the concentration ratio of Cu(II) and L_prolinamide can be explained by the shift of the equilibrium of Cu(II) +2(L-prolinamide) Cu(II)(L-prolinamide)2 to the formation of the corresponding complex.With the growth in the total concentration of complex Cu(II)(L-prolinamide)22+, the degree of binding of each of the enantiomers increases. Since the electrophoretic mobilities are the same for unbound enantiomers and are different for diastereomeric complexes, the increase in the fraction of the latter leads to an increasing difference in the electrophoretic mobilities of analytes and, consecutively, to an increase in the Rs value 17.In general, the composition of the working electrolyte for LECE is selected regarding the best resolution of analytes at a low electric current and a stable baseline.Chiral ligand-exchange capillary electrophoresis.Chiral separation using ligand-exchange capillary electrophoresis was performed for the first time in 1985 in the determination of the dansyl derivatives of amino acid enantiomers 3. D- and L-isomers were separated by adding the copper(II) salt and L-histidine into the working electrolyte. Two years later, the similar experiments were performed by the same research group with the use of aspartame as a chiral selector (Fig. 2) 4.Fig. 2. Structure of the complexes formed by aspartame and the dansyl amino acid with the Cu2+ ions 4.Further development of the method was aimed to search the versions of the electrophoretic separation of optically active isomers without derivatization. Similarly to ligand-exchange systems in chiral ligand-exchange chromatography 1, 2, 18, in further experiments on the separation of enantiomers by LECE, Cu2+ ions were used as a complexing metal, and amino acid L-proline and its derivatives, as an optically active ligand. It was shown that some derivatives of L-proline (for example, L-4-hydroxyproline) ensured higher separation selectivity 8, 9. However, this ligand is suitable only for the separation of the enantiomers of amino acids bearing an aromatic ring 8, 9.For more effective interaction with analytes, N- (2- hydroxypropyl)- L- 4- hydroxyproline (HP- L-HyPro) and N- (2-hydroxyoctyl)- L- 4- hydroxyproline (HO-L-HyPro) were proposed as chiral selectors (Fig. 3), capable of separating aliphatic acids as well as aromatic 9. It was shown that bound in the complex with Cu2+ ions, they exhibit higher enantioselectivity with respect to L-4-hydroxyproline. For example, only the enantiomers of histidine were managed to be separated (Rs = 1.84) among 25 amino acids with the use of L-4-hydroxyproline (L-HyPro). When L-HyPro is replaced by its derivatives HP-L-HyPro and HO-L-HyPro, the number of amino acids, whose enantiomers can be separated (Rs > 1.3), increases to 15 and 18, respectively. N- (2-Hydroxyoctyl)- L- 4- hydroxyproline is also used for separating aliphatic and aromatic -amino acids and amino alcohols by LECE 9.Fig. 3. Structure of N-(2-hydroxypropyl)-L-4-hydroxyproline (R = CH3) and N-(2-hydroxyoctyl)-L-4-hydroxyproline (R = C6H13).It was demonstrated that the presence of the hydrophobic group, the alkyl substituent (R2) interacting with the alkyl or aryl radical in the amino acid molecule (R1),has a stabilizing effect. The structures of the formed mixed-ligand diastereomeric complexes are proposed (Fig. 4).Fig. 4. Structure of the diastereomeric complexes of Cu(II) formed by the amino acid and the chiral selector, N-alkyl-L-4-hydroxyproline (R2 = CH3, C6H13) 8, 9.For the last two years, the results obtained by the Japanese scientific group have been published on the use of chiral zinc complexes with various ligands in LECE. This approach has a limitation that only analytes absorbing in the UV region can be determined 25-28.The combination of the ligand-exchange mechanism of the electrophoretic separation of analytes with the chromatographic mechanism, effectuated in micellar electrokinetic chromatography, allows the separation of optical, geometric, and position isomers which was demonstrated by the example of the amino acid derivatives 14, 29. Such an approach can be implemented by adding a complex of Cu2+ with a ligand, whose molecule contains a hydrophobic alkyl fragment, for example, N,N-didecyl-D-alanine, as a chiral selector into the working electrolyte, which was used for separating the dansyl amino acid derivatives 7. The analytes in the form of the mixed-ligand complexes with Cu2+ and N,N- didecyl- D- alanine are distributed between the hydrophobic micellar (pseudostationary) phase and the working electrolyte. This ligand can also be used as an addition into the mobile phase in the chromatographic separation of optical isomers 7.By introducing various functional groups into the cyclodextrin molecule, one can change the selectivity of the formation of the complex and affect the stabili