Enantiomeric separation of N‐protected amino acids by non‐aqueous capillary electrophoresis using quinine or Tert‐butyl carbamoylated quinine as chiral additive

A capillary electrophoretic (CE) method for the enantioseparation of N‐protected chiral amino acids was developed using quinine and tert‐butyl carbamoylated quinine as chiral selectors added to nonaqueous electrolyte solutions (NACE). A series of various N‐derivatized amino acids were tested as chiral selectands, and in order to optimize the CE enantioseparation of these compounds, different parameters were investigated: the nature of the organic solvent, the combination of different solvents, the nature and the concentration of the background electrolyte, the selector concentration, the capillary temperature, and the applied voltage. The influence of these factors on the separation of the analyte enantiomers and the electroosmotic flow was studied. Generally, with tert‐butyl carbamoylated quinine as chiral selector, better enantioseparations were achieved than with unmodified quinine. Optimum experimental conditions were found with a buffer made of 12.5 mM ammonia, 100 mM octanoic acid, and 10 mM tert‐butyl carbamoylated quinine in an ethanol–methanol mixture (60:40 v/v). Under these conditions, DNB‐Leu enantiomers could be separated with a selectivity factor (α) of 1.572 and a resolution (Rs) of 64.3; a plate number (N) of 127,000 and an asymmetry factor (As) of 0.93 were obtained for the first migrating enantiomer. Chirality 11:622–630, 1999. © 1999 Wiley‐Liss, Inc.     

Evaluation of the macrocyclic antibiotic LY333328 as a chiral selector when used as a mobile phase additive in narrow bore HPLC

The macrocyclic antibiotic LY333328 has been evaluated as a chiral selector for the enantioseparation of nine dansylated amino acids. This macrocyclic glycopeptide was used as a chiral mobile phase additive (CMPA) in conjunction with narrow bore high‐performance liquid chromatography (HPLC). The key mobile phase parameters of LY333328 concentration and buffer pH were varied, along with variations in stationary phases consisting of C8, phenyl, cyano, and silica. After observing and plotting changes in retention and resolution based on corresponding variation in these parameters, a better understanding of the behavior of this chiral selector was obtained. The pK_a values of the dansyl amino acid analytes and LY333328 were measured and used to gain a better understanding of the microenvironment in which these enantioseparations occur. Optimized conditions resulted in the baseline separation of eight of nine dansyl amino acids. Chirality 11:75–81, 1999. © 1999 Wiley‐Liss, Inc.     

Synergistic chiral separations using the glycopeptides ristocetin A and vancomycin

The effectiveness of using a mixture of the chiral selectors vancomycin and ristocetin A to achieve chiral recognition was examined in this study. The results of using the mixed chiral selector vancomycin and ristocetin A in capillary electrophoresis were compared with the results of using each chiral selector alone. Chiral separations were carried out using a coated capillary column to suppress electroosmotic flow and minimize interactions with the capillary wall. We employed a countercurrent process where the solute reaches the detection cell window after the chiral selector has cleared the window, minimizing the background absorbance from the chiral selector and improving sensitivity. Using a mixture of vancomycin and ristocetin A, separations were achieved which often exceeded the resolving power of either chiral selector when used alone. The effect of voltage on resolution was also studied, and the optimal voltage was found to be between -5 and -8 kV.     

Direct chiral separation of unnatural amino acids by high-performance liquid chromatography on a ristocetin a-bonded stationary phase.

Direct high-performance liquid chromatographic chiral separation of numerous underivatized unnatural amino acids on a ristocetin A-bonded chiral stationary phase used in the reversed-phase and in the polar organic chromatographic modes is reported. The effects of different parameters such as mobile phase composition, temperature, and the structure of the analytes on the selectivity in both chromatographic modes are discussed. By variation of the parameters, the separation of the stereoisomers was optimized and, as a result, baseline resolution was achieved in most cases.     

New data on the sorption properties of α1‐acid glycoprotein chiral stationary phase

A binding study on α₁‐acid glycoprotein (AGP), a widely used chiral stationary phase in drug analysis, has been performed. Being a selectivity decisive factor in enantiomer separation, the adsorption of the organic modifiers, as the regularly used acetonitrile (AcCN) and also of dioxane, was determined from phosphate buffer eluents (pH 7.2 and 4.0) in the concentration range of 0.57–3.81 M and 0.11–1.9 M, respectively. The adsorbed amount was determined by gas chromatography. At lower modifier concentrations no significant difference was found in the binding extent of AcCN and dioxane. At higher, characteristic concentrations of the organic additives, a saturation was obtained at both pH values; furthermore, at pH 4.0 it was followed by a definite rise in the modifier adsorption. This particular behaviour may indicate the exposure of new binding sites on AGP surface, as a result of changes in the protein structure, which was confirmed by CD‐spectroscopic measurements. The pH dependence of binding in equimolar concentration (1.9 M) has shown the priority of dioxane at pH 4.0, while the adsorption of AcCN dominated at higher pH, indicating the different character of the two solvents. The increased hydrogen bond formation should cause the preferred adsorption of dioxane at pH 4.0. Chirality 11:212–217, 1999. © 1999 Wiley‐Liss, Inc.     

Evaluation of the specific intermolecular interactions responsible for chiral selectivity for phenylalanine analogs in subcritical fluid chromatography

A set of phenyl ring‐substituted N‐t‐butoxycarbonyl‐phenylalanine analogs were chirally resolved using an α‐Burke 2 Pirkle‐type chiral column under subcritical fluid conditions. Various mobile phase modifiers were used to elute the chiral analytes, resulting in different selectivity factors for each analog. The observed selectivity factors were accurately modeled based on the bulk solvation parameters for each mobile phase modifier. The resulting model equation was used to predict the selectivity factors using an additional modifier not included in the model building data set. The predictive ability of the model was demonstrated to be quite good for this limited range of analogs and mobile phase modifiers. Chirality 11:98–102, 1999. © 1999 Wiley‐Liss, Inc.     

Investigation of the Enantioselectivity of Tetramethylammonium L‐hydroxyproline Ionic Liquid as a Novel Chiral Ligand in Ligand‐Exchange CE and Ligand‐Exchange MEKC

Chiral ionic liquids (ILs) have drawn more and more attention in separation science; however, only a few papers focused on the application of chiral ILs as chiral ligands in LE‐CE. In this article, a novel amino acid ionic liquid (AAIL), tetramethylammonium L‐hydroxyproline ([TMA][L‐OH‐Pro]), was first applied as a chiral ligand to evaluate its enantioselectivity towards several aromatic amino acids in ligand‐exchange capillary electrophoresis (LE‐CE) and ligand‐exchange micellar electrokinetic capillary chromatography (LE‐MEKC). In the LE‐CE system, excellent separations were achieved for tryptophan (Rs = 3.03) and 3, 4‐dihydroxyphenylalanine (DOPA) (Rs = 4.35). Several parameters affecting the enantioseparation were systematically investigated, including AAIL concentration, type and concentration of central metal ion, buffer pH, as well as applied voltage. The optimum separation was obtained with 60 mM AAIL containing 30 mM Cu (II) at pH 4.5. Additionally, an LE‐MEKC system was established to further study the enantioselectivity of [TMA][L‐OH‐Pro] towards selected analytes. As observed, the separations of the enantiomers of tryptophan, phenylalanine, and histidine were all improved compared to the LE‐CE system. The results indicated that the application of AAILs as chiral ligands is a promising method in chiral separation science. Chirality 27:58–63, 2015. © 2014 Wiley Periodicals, Inc.     

Enantioseparation of Racemic Flurbiprofen by Aqueous Two‐Phase Extraction With Binary Chiral Selectors of L‐dioctyl Tartrate and L‐tryptophan

A novel method for chiral separation of flurbiprofen enantiomers was developed using aqueous two‐phase extraction (ATPE) coupled with biphasic recognition chiral extraction (BRCE). An aqueous two‐phase system (ATPS) was used as an extracting solvent which was composed of ethanol (35.0% w/w) and ammonium sulfate (18.0% w/w). The chiral selectors in ATPS for BRCE consideration were L‐dioctyl tartrate and L‐tryptophan, which were screened from amino acids, β‐cyclodextrin derivatives, and L‐tartrate esters. Factors such as the amounts of L‐dioctyl tartrate and L‐tryptophan, pH, flurbiprofen concentration, and the operation temperature were investigated in terms of chiral separation of flurbiprofen enantiomers. The optimum conditions were as follows: L‐dioctyl tartrate, 80 mg; L‐tryptophan, 40 mg; pH, 4.0; flurbiprofen concentration, 0.10 mmol/L; and temperature, 25 °C. The maximum separation factor α for flurbiprofen enantiomers could reach 2.34. The mechanism of chiral separation of flurbiprofen enantiomers is discussed and studied. The results showed that synergistic extraction has been established by L‐dioctyl tartrate and L‐tryptophan, which enantioselectively recognized R‐ and S‐enantiomers in top and bottom phases, respectively. Compared to conventional liquid–liquid extraction, ATPE coupled with BRCE possessed higher separation efficiency and enantioselectivity without the use of any other organic solvents. The proposed method is a potential and powerful alternative to conventional extraction for separation of various enantiomers. Chirality 27:650–657, 2015. © 2015 Wiley Periodicals, Inc.     

Comparison of Chiral Separations of Aminophosphonic Acids and Their Aminocarboxylic Acid Analogs Using a Crown Ether Column

Crown ethers are capable of complexing with primary amines and have been utilized in chromatography to separate amino acid racemates. This application has been extended to resolve (1‐amino‐1‐phenylmethyl)phosphonic acid and (1‐aminoethyl)phosphonic acid racemates, along with their aminocarboxylic acid analogs (2‐phenylglycine and alanine, respectively), via a ChiroSil RCA crown ether based chiral stationary phase. Effects of the organic modifier, temperature, and acid type and concentration on retention and selectivity were also investigated. Trends in retention and selectivity varied between aminophosponic acids and their aminocarboxylic analogs. Computer modeling and ¹H NMR analyses were performed to potentially gain a better understanding of interactions of the aforementioned molecules with the ChiroSil RCA chiral stationary phase. Theoretical predictions of the most stable conformations for (R)‐ and (S)‐enantiomers were compared to elution order; it was found that the elution order agreed with molecular modeling such that the longest retention correlated with the predicted most stable complex between the enantiomer and crown ether. ¹H NMR demonstrated interactions of aminophosphonic and aminocarboxylic racemates with (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid in solution and was utilized to determine enantiomeric excess of (1‐amino‐1‐phenylmethyl)phosphonic acid after its enantioenrichment via crystallization through diastereomeric salt formation with the crown ether followed by filtration. Chirality 25:369–378, 2013. © 2013 Wiley Periodicals, Inc.     

Effect of acidic mobile phase additives on chiral selectivity for phenylalanine analogs using subcritical fluid chromatography

A variety of acidic mobile phase additives were investigated as to their effects on retention, selectivity, efficiency, and overall chiral resolution for a number of chiral N‐substituted phenylalanine analogs under subcritical conditions. These mobile phase additives showed significant effects for all of the chromatographic parameters evaluated in this study. All of the phenylalanine analogs showed decreasing retention as the pK_a of the additive decreased. Plots of selectivity, efficiency, and chiral resolution showed pronounced improvement using acidic additives with pK_a values near −1. These results demonstrated that the choice of acidic mobile phase additives had a significant effect on the resulting chromatography for these chiral analytes under subcritical conditions. Chirality 11:91–97, 1999. © 1999 Wiley‐Liss, Inc.     

Enantioselective resolution of 4‐chloromandelic acid by liquid–liquid extraction using 2‐chloro‐N‐carbobenzyloxy‐L‐amino acid

A liquid–liquid extraction resolution of 4‐chloro‐mandelic acid (4‐ClMA) was studied by using 2‐chloro‐N‐carbobenzyloxy‐L‐amino acid (2‐Cl‐Z‐AA) as a chiral extractant. Important factors affecting the extraction efficiency were investigated, including the type of chiral extractant, pH value of aqueous phase, initial concentration of chiral extractant in organic phase, initial concentration of 4‐ClMA in aqueous phase, and resolution temperature. It was observed that the concentration of (R)‐4‐ClMA was much higher than that of (S)‐4‐ClMA in organic phase due to a higher stability of the complex formed between (R)‐4‐ClMA and 2‐Cl‐Z‐AA. A separation factor (α) of 3.05 was obtained at 0.02 mol/L 2‐Cl‐Z‐Valine dissolved in dichloromethane, pH of 2.0, concentration of 4‐ClMA of 0.11 mmol/Land T of 296.7K.     

Enantiomeric separation of dansyl amino acids using macrocyclic antibiotics as chiral mobile phase additives by narrow-bore high-performance liquid chromatography

Seven macrocyclic antibiotics were evaluated as chiral selectors for the enantiomeric separation of 11 dansyl amino acids using narrow-bore high-performance liquid chromatography (HPLC). The macrocyclic antibiotics were incorporated as mobile phase additives to determine the enantioselective effects on the chiral analytes. The resolution and capacity factor (k') of each analyte were assessed while varying the structure of macrocyclic antibiotic and the mobile phase buffer pH. The selectivity of the chiral selectors was measured as a function of changes in these parameters. All 11 dansyl amino acids were separated by at least one of the chiral selectors. Three-dimensional computer modeling of the more effective chiral selectors illustrated the importance of macrocyclic antibiotic structure concerning stereospecific analyte interaction.     

Chiral separation of aliphatic primary amino alcohols as o‐phthaldialdehyde/mercaptoethanol derivatives on polysaccharide‐based chiral stationary phases

A sensitive chiral high performance liquid chromatography (HPLC) method for the determination of aliphatic primary amino alcohol isomers with o‐phthaldialdehyde/mercaptoethanol precolumn derivatization has been developed and validated. Seven chiral columns were tested in a reversed phase mode. Excellent enantioseparation with the resolution more than 2.0 was achieved on Chiralcel OJ‐3R. The effect of various chromatographic conditions including column temperature, acetonitrile content in the mobile phase, buffer pH, buffer concentration, and buffer type in the mobile phase on the retention and the selectivity was investigated. The final mobile phase consisted of binary mixture of 20mM ammonium formate solution with acetonitrile (75:25; v/v). The analyses were performed at mobile phase flow rate of 1.0 mL/min and the column temperature of 40°C. The fluorescence detection was performed at excitation wavelength of 345 nm and emission wavelength of 450 nm. The developed method was fully validated in terms of linearity, sensitivity, accuracy, precision, intermediate precision, and selectivity according to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines using internal normalization procedure. The proposed chiral method was proved to be highly sensitive, simple, and rapid and was successfully applied to the determination of D‐Valinol content in commercially available samples of L‐Valinol.     

Chiral Separation of the Clinically Important Compounds Fucose and Pipecolic Acid Using CE: Determination of the Most Effective Chiral Selector

In this study, simple electrophoretic methods were developed for the chiral separation of the clinically important compounds fucose and pipecolic acid. In recent years, these analytes, and particularly their individual enantiomers, have attracted considerable attention due to their role in biological functions and disorders. The detectability and sensitivity of pipecolic acid and fucose were improved by reacting them with fluorenylmethyloxycarbonyl chloride (FMOC‐Cl) and 5‐amino‐2‐naphthalene‐sulfonic acid (ANSA), respectively. The enantioseparation conditions were optimized by initially investigating the type of the chiral selector. Different chiral selectors, such as polymeric surfactants and cyclodextrins, were used and the most effective ones were determined with regard to resolution and analysis time. A 10‐mM β‐cyclodextrin was able to separate the enantiomers of ANSA‐DL‐fucose and the polymeric surfactant poly(sodium N‐undecanoyl‐LL‐leucine‐valinate) was able to separate the enantiomers of FMOC‐DL‐pipecolic acid, with resolution values of 3.45 and 2.78, respectively. Additional parameters, such as the concentration and the pH of the background electrolyte (BGE), the concentration of the chiral selector, and the addition of modifiers were examined in order to optimize the separations. The addition of the chiral ionic liquid D‐alanine tert‐butyl ester lactate into the BGE was also investigated, for the first time, in order to improve resolution of the enantiomers. Chirality 25:556–560, 2013. © 2013 Wiley Periodicals, Inc.     

Study of the stability of promethazine enantiomers by liquid chromatography using a vancomycin-bonded chiral stationary phase

Three chiral stationary phases based on macrocyclic antibiotics (teicoplanin, vancomycin and ristocetin A) have been tested for chiral separations of promethazine. The vancomycin phase permits the best, baseline enantioseparation of promethazine, with a mobile phase of a 80:20 (v/v) mixture of methanol with a 1% aqueous triethylamine acetate buffer of pH 4.1 and with the analysis time not exceeding 15 min. The limits of detection amount to 27.5 and 31.0 ng/ml for the earlier and later eluting enantiomers, respectively. This separation system, that also permits a sufficient resolution between the promethazine enantiomers and their degradation products, has further been used for the monitoring of the effects of light, temperature and the promethazine concentration in solution on the stability of methanolic promethazine solutions over a period of 19 days. It has been found that the stability of more concentrated solutions is primarily affected by the temperature, whereas the effects of the temperature and light are comparable with more dilute solutions. After 19 days, a solution of 0.5 mg/ml promethazine stored in darkness at a low temperature still contained 84.0% of the original amount of the enantiomers; this value was 89.6% for a solution with the ten times lower promethazine concentration. If the solutions were stored in darkness but at laboratory temperature, the respective values decreased to 38.1 and 62.6% and for the solutions exposed to light at laboratory temperature they decreased even more to 36.7 and 52.6% of the initial promethazine amount.     

Enantiomeric resolution of some nonsteroidal antiinflammatory and anticoagulant drugs using β‐cyclodextrins by capillary electrophoresis

β‐cyclodextrin (CD) and its derivatives HP‐β‐CD, DM‐β‐CD, and TM‐β‐CD have been employed as chiral selectors for the separation of three nonsteroidal antiinflammatory drugs (NSAIDs) and anticoagulant at relatively low concentration (8–15 mM) by capillary zone electrophoresis (CZE). In this study, baseline separation was achieved for ibuprofen, ketoprofen, naproxen, and warfarin. It was found that the addition of 0.1% hydroxypropyl methyl cellulose (HPMC) was effective for separation. Under these conditions, the S‐(+) enantiomer eluted before R‐(−) in terms of ibuprofen; the calculated energy values obtained from the molecular modeling correlated well with the elution order. An equation for calculating the pK_a values by capillary electrophoresis was introduced, and the pK_a values of the four chiral drugs at 25°C were obtained based on the equation. The value pK_a + 0.5 is proposed to be the suitable pH of the background electrolyte for the separation of chiral compounds containing a carboxylic group. Chirality 11:56–62, 1999. © 1999 Wiley‐Liss, Inc.     

Evaluation of chiral separation efficiency of a novel OTPTHE derivatization reagent: Applications to liquid‐chromatographic determination of DL‐serine in human plasma

A novel chiral derivatization reagent, the N‐[1‐oxo‐5‐(triphenylphosphonium)pentyl]‐ (R)‐1,3‐thiazolidinyl‐4‐N‐hydroxysuccinimide ester bromide salt (OTPTHE), was developed for the separation and selective detection of chiral DL‐amino acids by RP‐HPLC analysis. The OTPTHE reacted with DL‐amino acids at 60°C maintained for 30 minutes in the presence of 100 mM borate buffer (pH 9.5). The separability of the diastereomeric derivatives was evaluated in terms of the resolution value (Rs) using 13 kinds of DL‐amino acids, which were completely separated by reversed‐phase chromatography using C18 column at 254 nm. The Rs of the DL‐amino acids varied from 1.62 to 2.51. As for the application of the DL‐amino acids, the determination of DL‐Ser in the human plasma of healthy volunteers was performed based on our developed method. It was shown that linear calibrations were available with high coefficients of correlation (r² > 0.9997). The limit of detection (S/N = 3) of the DL‐Ser enantiomers was 5.0 pmol; the relative standard deviations of the intraday and interday variations were below 4.56%; the accuracy ranged between 95.40%‐110.06% and 95.45%‐109.80%, respectively; the mean recoveries (%) of the DL‐Ser spiked in the human plasma were 99.49%‐103.74%. The amounts of DL‐Ser in the human plasma of healthy volunteers were determined.     

Investigation of maltodextrin‐based synergistic system with amino acid chiral ionic liquid as additive for enantioseparation in capillary electrophoresis

The combined use of chiral ionic liquids (ILs) and chiral selectors in capillary electrophoresis (CE) to establish a synergistic system has proven to be an effective approach for enantioseparation. In this article, tetramethylammonium‐L‐arginine, a kind of amino acid chiral IL, was applied to investigate its potential synergistic effect with maltodextrin in CE enantioseparation. The established maltodextrin‐based synergistic system showed markedly improved enantioseparations compared with the single maltodextrin system. Parameters such as the chiral IL concentration, maltodextrin concentration, buffer pH, applied voltage, and capillary temperature were optimized. Satisfactory enantioseparation of the five studied drugs, including nefopam, duloxetine, ketoconazole, cetirizine, and citalopram was achieved in 50 mM Tris‐H₃PO₄ buffer solution (pH 3.0) containing 7.0% (m/v) maltodextrin and 60 mM tetramethylammonium‐L‐arginine. In addition, the chiral configuration of tetramethylammonium‐L‐arginine was also investigated to demonstrate the existence of a synergistic effect between chiral ILs and maltodextrin.     

Chiral Separation of Four Stereoisomers of Ketoconazole Drugs Using Capillary Electrophoresis

This work aimed to develop a chiral separation method of ketoconazole enantiomers using electrokinetic chromatography. The separation was achieved using heptakis (2, 3, 6‐tri‐O‐methyl)‐β‐cyclodextrin (TMβCD), a commonly used chiral selector (CS), as it is relatively inexpensive and has a low UV absorbance in addition to an anionic surfactant, sodium dodecyl sulfate (SDS). The influence of TMβCD concentration, phosphate buffer concentration, SDS concentration, buffer pH, and applied voltage were investigated. The optimum conditions for chiral separation of ketoconazole was achieved using 10 mM phosphate buffer at pH 2.5 containing 20 mM TMβCD, 5 mM SDS, and 1.0% (v/v) methanol with an applied voltage of 25 kV at 25 °C with a 5‐s injection time (hydrodynamic injection). The four ketoconazole stereoisomers were successfully resolved for the first time within 17 min (total analysis time was 28 min including capillary conditioning). The migration time precision of this method was examined to give repeatability and reproducibility with RSDs ≤5.80% (n =3) and RSDs ≤8.88% (n =9), respectively. Chirality 27:223–227, 2015. © 2014 Wiley Periodicals, Inc.     

Selective separation of amino acids with a charged inorganic nanofiltration membrane: effect of physicochemical parameters on selectivity

A charged organic-inorganic nanofiltration (NF) membrane prototype was used to separate a mixture of nine amino acids (AA) on the basis of differential electrostatic interactions with the membrane because, for a given pH, some of them were positively charged, some were negative, and some were zwitterions. Effect of pH, amino acid concentration (C(r)), and added ionic strength ([NaCI]) on the process selectivity was studied. A global statistical study revealed that pH was the dominant parameter regarding fractionation. C(r) and [NaCI] had a weaker effect, but the ratio C(r)/[NaCI] demonstrated a pronounced effect on system selectivity. Two split-ups of the mixture were obtained at pH 2 and at pH 12, for a 1-g/L total AA concentration and a C(r)/[NaCI] ratio of 0.16. Under these conditions, the differences in transmissions between basic and acid AA were higher than 70%. Interpretation of the results according to the Donnan theory allows us to foresee the potentialities of charged nanofiltration membranes for the fractionation of a complex mixture, such as peptidic hydrolysate to streams containing peptides and amino acids having different isoelectric points. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 291-302, 1997.