Use of a Novel Sub‐2 µm Silica Hydride Vancomycin Stationary Phase in Nano‐Liquid Chromatography. II. Separation of Derivatized Amino Acid Enantiomers

A novel vancomycin silica hydride stationary phase was synthesized and the particles of 1.8 µm were packed into fused silica capillaries of 75 µm internal diameter (I.D.). The chiral stationary phase (CSP) was tested for the separation of some derivatized amino acid enantiomers by using nano‐liquid chromatography (nano‐LC). Some experimental parameters such as the type and the content of organic modifier, the pH, and the concentration of the buffer added to the mobile phase were modified and the effect on enantioselectivity, retention time, and enantioresolution factor was studied. The separation of selected dansyl amino acids (Dns‐AAs), e.g., Asp, Glu, Leu, and Phe in their enantiomers was initially achieved utilizing a mobile phase containing 85% (v/v) methanol (MeOH) and formate buffer measuring the enantioresolution factor and enantioselectivity in the range 1.74–4.17 and 1.39–1.59, respectively. Better results were obtained employing a more polar organic solvent as acetonitrile (ACN) in the mobile phase. Optimum results (Rs 1.41–6.09 and α 1.28–2.36) were obtained using a mobile phase containing formate buffer pH 2.5/water/MeOH/ACN 6:19:12.5:62.5 (v/v/v/v) in isocratic elution mode at flow rate of 130 nL/min. Chirality 27:767–772, 2015. © 2015 Wiley Periodicals, Inc.     

Chiral recognition ability and solvent versatility of bonded amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase: enantioselective liquid chromatographic resolution of racemic N-alkylated barbiturates and thalidomide analogs

The chiral recognition ability and solvent versatility of a new chiral stationary phase containing amylose 3,5-dimethylphenylcarabamate immobilized onto silica gel (CHIRALPAK IA) is investigated. Thus, the direct enantioselective separation of a set of racemic N-alkylated barbiturates and 3-alkylated analogs of thalidomide was conducted using different nonstandard solvents as eluent and diluent, respectively in high-performance liquid chromatography (HPLC). The separation, resolution, and elution order of the investigated compounds were compared on both immobilized and coated amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phases (Chiralpak IA and Chiralpak AD, respectively) using a mixture of n-hexane/2-propanol (90:10 v/v) as mobile phase with different flow-rates and fixed UV detection at 254 nm. The effect of the immobilization of the amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase on silica (Chiralpak IA) on the chiral recognition ability was noted as the bonded phase (Chiralpak IA) was superior in chiral recognition and possesses a higher resolving power in most of the reported cases than the coated one (Chiralpak AD). A few racemates were not or poorly resolved on the immobilized Chiralpak IA or the coated Chiralpak AD when using standard solvents were most efficiently resolved on the immobilized Chiralpak IA upon using nonstandard solvents. Furthermore, the immobilized phase withstands the nonstandard (prohibited) HPLC solvents such as dichloromethane, ethyl acetate, tetrahydrofuran, methyl-tert-butyl ether, and others when used as eluents or as a dissolving agent for the analyte itself. The direct analysis of a real sample extracted from plasma using DCM on Chiralpak IA is also shown.     

Optimization of separation of purine and pyrimidine compounds using ion-pair reversed-phase high performance chromatography (HPLC)

Utilization of ion-air reagents in a reversed-phase chromatographic system allows solving a number of problems related to the separation of purine and pyrimidine derivatives. Simultaneous analysis of nucleotides, nucleosides and their bases was carried out by acetonitrile gradient elution using tetrabutyl ammonium phosphate as a counterion in the mobile phase. Besides, optimal conditions were selected for isocratic separation of adenine nucleotides and their metabolites. Furthermore, isocratic separation of certain purines and pyrimidines was achieved by modifying the stationary C18-phase with pentane- and heptane sulphonic acids.     

Resolution,determination of enantiomeric purity and chiral recognition mechanism of new xanthone derivatives on (S,S)‐whelk‐O1 stationary phase

The enantioresolution and determination of the enantiomeric purity of 32 new xanthone derivatives, synthesized in enantiomerically pure form, were investigated on (S ,S )‐Whelk‐O1 chiral stationary phase (CSP). Enantioselectivity and resolution (α and R_S) with values ranging from 1.41–6.25 and from 1.29–17.20, respectively, were achieved. The elution was in polar organic mode with acetonitrile/methanol (50:50 v/v ) as mobile phase and, generally, the (R )‐enantiomer was the first to elute. The enantiomeric excess (ee ) for all synthesized xanthone derivatives was higher than 99%. All the enantiomeric pairs were enantioseparated, even those without an aromatic moiety linked to the stereogenic center. Computational studies for molecular docking were carried out to perform a qualitative analysis of the enantioresolution and to explore the chiral recognition mechanisms. The in silico results were consistent with the chromatographic parameters and elution orders. The interactions between the CSP and the xanthone derivatives involved in the chromatographic enantioseparation were elucidated.     

High pressure liquid chromatographic identification of hyaluronic acid and chondroitin sulphate disaccharides.

In this report we describe a system capable of resolving all of the known unsaturated disaccharides derived from the chondroitin sulphates, dermatan sulphate and hyaluronic acid by chondroitinase digestion. This system is superior to others in that the non-sulphated and mono-, di- and tri-sulphated disaccharides can be separated with good resolution in approximately 40 min in an isocratic solvent. The system employs an amino-cyano silica gel column (Whatman Partisil 5 PAC, 25 cm) and is eluted with an isocratic solvent consisting of 48% (v/v) acetonitrile, 14% (v/v) methanol and 38% (v/v) aqueous buffer. This aqueous buffer contains 0.5 M Tris-HCl, 0.1 M boric acid, 23.4 mM sulphuric acid, pH 8.0. UV absorption is monitored at 229 nm and for most disaccharides as little as 150 ng can be reliably determined. The addition of boric acid to the eluent is essential for good resolution of all components and the addition of low concentrations of sulphuric acid is used to control the elution times of various components. The system was applied to the analysis of glycosaminoglycan standards and excellent agreement with previous compositional analyses was obtained.     

Enantioseparation and molecular modeling study of chiral amines as three naphthaldimine derivatives using amylose or cellulose trisphenylcarbamate chiral stationary phases

This study describes the enantioseparation of three chiral amines as naphthaldimine derivatives, using normal phase HPLC with amylose and cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phases (CSPs). Three chiral amines were derivatized using three structurally similar naphthaldehyde derivatizing agents, and the enantioselectivity of the CSPs toward the derivatives was examined. The degree of enantioseparation and resolution was affected by the amylose or cellulose-derived CSPs and aromatic moieties as well as a kind of chiral amine. Especially, efficient enantiomer separation was observed for 2-hydroxynapthaldimine derivatives on cellulose-derived CSPs. Molecular docking studies of three naphthaldimine derivatives of leucinol on cellulose tris(3,5-dimethylphenylcarbamate) were performed to estimate the binding energies and conformations of the CSP–analyte complexes. The obtained binding energies were in good agreement with the experimentally determined enantioseparation and elution order.     

Polyethylene-based high-performance liquid chromatography of chloroplast pigments: resolution of mono- and divinyl chlorophyllides and other pigment mixtures

In addition to most chlorophylls and their derivatives, monovinyl and divinyl chlorophyll species were separated by high-performance liquid chromatography, using a polyethylene column and a simple elution with aqueous acetone. Peak retention and resolution of the pigment separation were greatly increased by increasing the polarity of the mobile phase and also by decreasing the column temperature. Polyethylene chromatography showed chlorophyll separation behavior similar to that of the octadecyl silica column, but it showed no adsorption of the pigment species containing free carboxylic acid groups, enabling the complete separation of chlorophylls and their derivatives. Polyethylene is a superior alternative stationary phase to the known reversed-phase materials for chlorophyll separation and analysis.     

High-performance liquid chromatographic resolution of (R,S)-α-alkyl-α-amino acids as diastereomeric derivatives

Summary A number (27) of racemic-alkyl--amino acids (AAA) were derivatized either witho-phthaldialdehyde (OPA) in combination withN-t-butoxycarbonyl-L-cysteine (Boc-Cys) orN-acetyl-L-cysteine (Ac-Cys), or withN ²-(5-fluoro-2,4-dinitrophenyl)-L-alanine amide (Marfey's reagent). The resolution of the diastereoisomers formed was investigated by reversed-phase (C₁₈) high-performance liquid chromatography (HPLC) using gradient elution conditions employing sodium phosphate buffers of pH 7.2 together with acetonitrile, and fluorescence detection at 344 nm (excitation) and 443 nm (emission) for the OPA/Boc-Cys or OPA/Ac-Cys derivatives. For the diastereomers formed by derivatization with Marfey's reagent triethylammonium phosphate buffers of pH 3.0 (pH 7.2 for acidic AAA) together with acetonitrile, and u.v. detection at 340 nm were used. Whereas with Marfey's reagent all diastereomers of AAA showed complete, or almost complete, resolution, only 8, or 11, respectively of the diastereomers formed by derivatization with OPA/Boc-Cys or OPA/Ac-Cys were resolved under the chromatographic conditions used.     

High-performance liquid chromatographic enantioseparation of N-protected α-amino acids using nonporous silica modified by a quinine carbamate as chiral stationary phase

In this study, tert-butyl carbamoylated quinine as chiral selector was immobilized on nonporous silica (NPS) 1.5 μm particles developed by MICRA, and this new chiral stationary phase (CSP) was packed into a 3.3 cm column (4.6 mm ID). A series of various N-protected α-amino acids was chosen as chiral selectands, including 3.5-dinitrobenzyloxycarbonyl amino acids (DNZ-AAs). In order to optimize the chromatographic conditions with this novel CSP and to apply it to the resolution of acidic analytes the following parameters have been varied and studied: pH of the mobile phase, buffer concentration, and percentage of methanol or acetonitrile in the mobile phase. DryLab^R software was applied to optimize enantioseparation by simulating chromatographic functions of experimental conditions for isocratic and/or gradient runs. Thus, we were able to resolve a set of test compounds within several minutes, whereby our attention was particularly drawn to the resolution of DNZ-AA derivatives. Chirality 9:157–161, 1997. © 1997 Wiley-Liss, Inc.     

Reproducibility of gel-chromatography

We think it worthwhile to other experimenters to mention the following observations. Recently it was noted that gel chromatographic separations of the five fragments resulting from the degradation of cytochrome c by bromocyanogen became dependent on the batch number of the gel. This dependency concerns only separations of unprotected fragments, using 7% (v/v) formic acid as the eluent. The elution profile of protected compounds, which in turn run in 50% (v/v) formic acid, was unaffected.     

乙醇系统HPLC分析PTC－氨基酸的条件优化

首次报道用乙醇系统分析ＰＴＣ－氨基酸的新方法中各衍生物获得最佳分离的建立过程。ＰＴＣ－氨基酸衍生后溶于Ａ溶液,然后进样于４μｍＮｏｖａＰａｋＣ１８柱（３．９ｍｍ×１５０ｍｍ）。系统的优化步序包括全面调控流动相的ｐＨ值与ＴＥＡ浓度、乙醇梯度程序、柱温等诸多影响ＨＰＬＣ色谱行为的因素。最适条件为：Ａ溶液含０．１４ＭＧ酸钠、０．７５ｍｌ／ＬＴＥＡ、ＰＨ６．３５;Ｂ溶液为１００％乙醇;柱温３０℃。通过优化的乙醇梯度最终在约４４ｍｉｎ内将１５种ＰＴＣ－氨基酸很好地分离。此法可用于替代代表新科技水平的ＰＴＣ－氨基酸乙腈分析系统。     

Enantiomeric separation of mineralocorticoid receptor (hMR) antagonists using the Chiralcel OJ-H HPLC column with novel polar cosolvent eluent systems

This study demonstrates the increased versatility of the Chiralcel OJ-H stationary phase when using various alcohol/acetonitrile mobile phases. This chiral stationary phase has traditionally been employed in the normal phase mode and more recently with neat alcohols as eluents. Selected isomeric human mineralocorticoid receptor (hMR) antagonist pharmaceutical candidates and synthetic intermediates were separated using the Chiralcel OJ-H HPLC column with novel polar cosolvent eluent systems. The capacity factors, resolution, and selectivity of the chiral separations were assessed while varying the alcohol/acetonitrile composition and alcohol identity. The mixed polar eluents provide separations that are nearly always superior to both the traditional hexane-rich and single-alcohol "polar organic" eluents for the compounds tested in this article.     

Structural analysis of the xyloglucan from Phaseolus coccineus cell-walls using cellulase-derived oligosaccharides

Oligosaccharides, obtained by digestion of a xyloglucan from the cell walls of Phaseolus coccineus with cellulase, have been isolated by gel filtration. Oligosaccharides containing 2–6 residues accounted for ～57% of the hydrolysate, with larger oligosaccharides (d.p. ～10) and partially degraded xyloglucan accounting for ～32% of the polymer. The major glycosidic linkages were determined by methylation analysis. Methylated penta- and hexa-saccharide alditols were isolated by reverse-phase h.p.l.c. and characterised by e.i.-m.s. and f.a.b.-m.s. Methylated derivatives of the di-, tri-, and tetra-saccharide alditols were examined by g.l.c.-m.s. in the e.i. and c.i. modes. A structure based on these results is proposed.     

High-pressure liquid chromatographic separation of a mixture of corticosteroids, androgens, and progestins

Fifteen steroids including corticosteroids, androgens, progestins, and their derivatives were completely separated by reverse-phase high-pressure liquid chromatography on a ChemcoPak 7 ODS-H column in 50 min. The elution procedures were first with water:methanol:acetonitrile:isopropanol 55:32:6.5:7.5 (v/v) for 15 min and followed with a linear gradient elution for 35 min from 0 to 80% of water:methanol:n-butanol 40:40:20 (v/v). The applicability of this method was successfully demonstrated in the analyses of the biological samples of carp plasma, testis, and head kidney.     

Chiral-recognition chromatography of β-blockers on continuous polymer beds with immobilized cellulase as enantioselective protein

Columns prepared by coupling cellulase as a chiral selector to silica beads are very efficient for the separation of enantiomers. In this paper we show that continuous polymer beds compete favorably with silica beads as chromatographic supports for such separations. The chiral stationary phase is prepared either by entrapment in and simultaneous covalent linkage of ally1 cellulase to the continuous beds during their preparation or by covalent immobilization of cellulase on an epoxy-activated continuous bed. Enantiomers of β-blockers were separated rapidly and with high resolution. The enantiomers of practolol were thus baseline resolved within 45 sec. The recognition center–or at least part of it—coincides with the active center of the enzyme, since the enantiomers could not be separated in the presence of the competitive enzyme inhibitors cellobiose and D-glucose and the separation was also impaired upon addition of the substrate carboxymethyl cellulose to the eluent. Similar observations have been reported for silica columns derivatized with cellulase. The capacity factor and the separation selectivity could be tuned by the pH and the concentration of the mobile phase, a phosphate buffer. No modifier was required, as is sometimes the case with silica-based supports. The continuous beds give faster enantiomer separations than do columns of silica and are more pH-stable and cost effective to prepare. © 1993 Wiley-Liss, Inc.     

Theory-guided efficient strategy to maximize speed and resolution in rapid gradient LC-MS/MS bioanalysis

Reversed-phase gradient LC-MS/MS bioanalytical methods of 5-100% organic solvent in a 1-3 min gradient time are common in today's bioanalytical laboratory. The goal of this work was to develop a theory-guided systematic strategy for maximizing resolution and speed in rapid gradient LC-MS/MS bioanalysis. We studied the effect of gradient time (t(G)), initial and final eluent strength (% B=% organic), and flow rate (F) on the separation of multiple critical pairs (R(s)) and peak capacity (n(c)) in a gradient elution of a mixture of five structurally related compounds. By optimizing the gradient time t(G), the initial and final percentages of the organic component of the mobile phase, comparable resolution and peak capacity could be achieved in a shorter run time. More importantly, we demonstrated that higher flow rates improved resolution, peak capacity and reduced run time in rapid gradient separations on a 5 μm particle column. A straightforward mathematical explanation of the phenomenon was provided applying basic resolution equations in gradient elution theory. A systematic approach to execute a rapid gradient LC-MS/MS bioanalytical method to shorten run time and improve resolution is proposed, taking into consideration not only the analytes of interest but also potential matrix effects from the dosing vehicle and biological matrix.     

Supercritical fluid chromatography comparison of the poly(trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column with several derivatized polysaccharide-based stationary phases

The poly(trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column has so far been primarily used with normal phase and polar organic mobile phase chromatography. Its use in supercritical fluid chromatography (SFC) was investigated via the analysis of 40 commercial and 100 proprietary compounds using a 12-min gradient with methanol as a modifier. Results were then compared against those obtained from the popular derivatized polysaccharide-based chiral stationary phases (CSPs) such as Chiralpak AD-H and Chiralpak AS-H as well as Chiralcel OD-H and Chiralcel OJ-H columns. P-CAP demonstrated separation of 25% of the 140 total compounds, while each of the derivatized polysaccharide-based CSPs separated at least 46%. A study that compared the loading of 1,1'-bi-2-naphthol with P-CAP and Chiralpak AS columns indicated a similar trend in resolution vs. amount injected, though AS appeared capable of allowing a greater loading of material. The P-CAP column was found to be beneficial in the separation of a complex mixture of enantiomers and achiral impurities, where the derivatized polysaccharide-based columns did not show as desirable of a separation. A key advantage of this type of chiral stationary phase is the fact that it is available in both enantiomeric forms, allowing manipulation of elution order of enantiomers, which is especially helpful for preparative applications. P-CAP also demonstrated that it could resolve an achiral impurity from the desired compound in a different mixture, while the same impurity co-eluted on the Chiralpak AD-H column. Overall, the synthetic polymer-based P-CAP showed less chiral discrimination power compared to the derivatized polysaccharide-based CSPs under the conditions explored in this study.     

Chiral separation and modeling of baclofen,bupropion, and etodolac profens on amylose reversed phase chiral column

Chiral resolution of baclofen, bupropion, and etodolac profens was obtained with amylose derivatized chiral reversed stationary phase (carbamate groups). The eluent used for bupropion and etodolac was MeOH–water (20:80, v /v) and for baclofen was water–methanol (95:5, v /v). The eluent run rates, finding wavelength and temperature, were 1.0 mL/min, 220 nm and 27 ± 1 °C for all the eluents. The magnitude of the retardation factors for S‐ and R‐enantiomers of baclofen, bupropion, and etodolac were 1.37, 2.62, 2.25, 3.25, 1.8, and 3.0. The magnitudes of separation and resolution factors were 1.90, 1.44, and 1.67 and 2.77, 2.35, and 2.04. Limits of detection and quantitation were 1.0–2.0 and 5.1–10.0 μg/mL. Chiral recognition mechanisms were recognized by simulation and high‐performance liquid chromatography (HPLC) experiments. It was seen that hydrogen interactions, hydrophobic interactions, and π–π exchanges were the chief interactions for chiral recognition mechanisms. The described methods may be exploited for the chiral separation of baclofen, bupropion, and etodolac profens in any unknown sample.     

Postnatal hypoglycaemia and gluconeogenesis in the newborn rat. Delayed onset of gluconeogenesis in prematurely delivered newborns.

A normal-phase high-pressure liquid-chromatography system was used with amino-propyl-bonded silica as the column packing in order to resolve amino acids, reduced amino acid derivatives and reduced cross-links of collagen. The method utilized a solvent system comprising acetonitrile and 10 mM-potassium phosphate buffer, pH 4.3, as described by Schuster [(1980) Anal. Chem. 52, 617-620]. With modifications to the original gradient elution it was possible to resolve fully the radiolabelled components of reduced collagen in one run of less than 80 min. The method is very sensitive, and small biopsy samples can readily be investigated. Although solute retention times gradually decreased with repeated use, little loss of resolution of the reducible cross-links of collagen occurred during a 30-day trial period.     

High performance liquid chromatography of peptide bioregulators, their fragments and derivatives. II. Sorption of peptides from aqueous-organic eluents on octadecylsilylsilica gel and unmodified silica gel

Dependence of the peptide retention upon the organic component concentration in eluent has been studied. A parabolic dependence has been found in a wide range of acetonitrile concentrations. The effect observed with ODS- and unmodified silica as stationary phases extends analytical and preparative potentialities of HPLC of peptides.