Effects of chromatography conditions on intact protein separations for top-down proteomics

A chiral stationary phase (CSP) has been prepared by chemically bonding a chiral pseudo-18-crown-6 type host having a 1-phenyl-1,2-cyclohexanediol unit to 3-aminopropyl silica gel. The chiral column was prepared by the slurry-packing method in a stainless steel HPLC column. Normal mobile phases can be used with this CSP in contrast to conventional dynamic coating type CSPs. Enantiomers of 20 out of 30 amino compounds, including 20 amino acids, 2 amino acid methyl esters, 6 amino alcohols, and 2 lipophilic amines, were efficiently separated on columns with this CSP. It is noteworthy that 15 amino compounds out of 30 were separated with better separation factors and shorter retention times compared to the corresponding CSP having pseudo-18-crown-6 with 1-phenyl-1,2-ethanediol as a chiral unit. In view of the correlation between the enantiomer selectivities observed in chromatography and those obtained in gas phase FABMS-EL methods and solution phase titrations, chiral recognition in the host-guest interaction likely contributes to enantiomer separation.     

Preparation and evaluation of a chiral stationary phase covalently bound with a chiral pseudo-18-crown-6 ether having a phenolic hydroxy group for enantiomer separation of amino compounds

In order to develop a chiral stationary phase (CSP), which has even higher separation ability than the corresponding commercially available crown ether based CSP (OA-8000 having a pseudo-18-crown-6 ether with an OMe group as a selector), chemically bonded type CSP having a phenolic OH group on a crown ring was developed. Normal mobile phases with or without acid additive can be used with this OH type CSP in contrast to the conventional OMe type CSP which has a neutral chiral selector. Enantiomers of 25 out of 27 amino compounds, including 20 amino acids, 5 amino alcohols, and 2 lipophilic amines, were efficiently separated on a column with this CSP. Nine amino compounds out of 27 were separated with better separation factors than the corresponding OMe type CSP. It is noteworthy that the chromatography on this CSP exhibited excellent enantiomer-separations for amines and amino alcohols when triethyl amine was used as an additive in the mobile phase. Comparison of enantiomer separation ability on this OH type of CSP and on the OMe type of CSP and correlation between the enantioselectivity in chiral chromatography and that of the corresponding model compounds in solution imply that the chiral separation arose from chiral recognition in host guest interactions.     

Gas Chromatographic Separation of Enantiomers on Optically Active Metal-Complex-Free Stationary Phases. New Analytical Methods (24)

If a stationary phase A employed in gas chromatography possesses a chemical affinity for substance B, which is to be separated, then the retention behavior is not only determined by the normal physical equilibrium between the gas and liquid phases but also by the chemical equilibrium A + B ? AB. If A and B are chiral and A is present in optically active form while B is a racemic mixture, then it is possible to achieve a gas chromatographic enantiomer resolution without the isolation of diastereomers: the energetically different diastereomeric associates A_R B_R and A_R B_S are formed rapidly and reversibly. This enantiospecific resolution principle was first demonstrated in 1966 by the quantitative resolution of racemic amino acid derivatives on optically active peptide phases in analogy to the well-known stereospecificity of enzymes. The anchoring of the chiral resolving agent to thermally stable polysiloxanes together with the employment of high resolution capillary columns and the use of appropriate derivatization strategies has led to the development of enantiomer resolution into a routine modern method for many classes of substances. The demonstration of enantiospecificity in the gas chromatographic separation process is of fundamental interest, and its systematic study can result in a significant contribution to the understanding of the molecular mechanism of “chiral recognition”. The gas chromatographic separation of enantiomers has also proven to be an accurate and sensitive method for the determination of the enantiomeric composition of natural products and products of enantioselective transformations (asymmetric syntheses, “chiral pool” transformations, kinetic resolutions, biomimetic reactions) and for the quantification of racemization, e.g. in the synthesis and hydrolysis of peptides. In any research program devoted to the phenomenon of chirality, the gas chromatographic separation of the enantiomers of volatile compounds constitutes an indispensable modern instrumental technique.     

Cellulose Derivatives Used as Chiral Stationary Phases in Capillary Gas Chromatography

Abstract

In this work, we present the first separation of enantiomers in gas chromatography (GC) using a fused‐silica capillary column containing cellulose triacetate, cellulose triphenylcarbamate, or cellulose tris(3,5‐dimethylphenylcarbamate) as the new chiral stationary phase. The separated solutes included alcohols, amine, ketone, ether, ester, and amino acid. Their column efficiency, polarity, and chiral selectivity were studied. The retention mechanism was discussed. The results showed that those derivatives had relatively high chiral recognition abilities and can be used as the chiral stationary phases in GC.     

Enantiomer separation of pharmaceuticals by capillary gas chromatography with novel chiral stationary phases

New chiral stationary phases of polydimethylsiloxane anchored with (S)-(-)-t-leucine derivatives were provided for use in enantiomer separation of pharmaceuticals by capillary gas chromatography. Fifteen pharmaceuticals were separated into their enantiomeric pairs by converting them into pentafluoropropionyl and heptafluorobutyryl derivatives. The separation factor and resolution obtained from the new phases were superior to those from the conventional Chirasil-Val capillary column.     

Cyclodextrins as chiral stationary phases in capillary gas chromatography. Part II: Heptakis(3-O-acetyl-2,6-di-O-pentyl)-ß-cyclodextrin

Heptakis (3-O-acetyl-2,6-di-O-pentyl)-ß-cyclodextrin is used as a chiral stationary phase in capillary gas chromatography. High enantioselectivity towards trifluoro-acetylated α and β-chiral amines, amino alcohols, α- and β-amino acid esters, and cyclic trans-diols is observed. In contrast to chiral polysiloxane phases, where hydrogen bonding interaction is essential for enantiomer separation, in cyclodextrins inclusion properties contribute to enantioselectivity. This can be concluded from the separation of N-alkylated amino compounds. The new chiral stationary phase exhibits a wide operating temperature range and is stable above 200°C.     

环糊精衍生物气相色谱手性固定相研究进展

评述近年环糊精衍生物气相色谱手性固定相的研究进展,对环糊精衍生物进行分类并总结了近年来其在GC手性分离上的应用,介绍环糊精衍生物的手性分离机制及纯度问题进展,展望环糊精衍生物作GC手性固定相的应用前景。     

Recent development trends for chiral stationary phases based on chitosan derivatives,cyclofructan derivatives and chiral porous materials in high performance liquid chromatography

The separation of enantiomers by chromatographic methods, such as gas chromatography, high‐performance liquid chromatography and capillary electrochromatography, has become an increasingly significant challenge over the past few decades due to the demand of pharmaceutical, agrochemical, and food analysis. Among these chromatographic resolution methods, high‐performance liquid chromatography based on chiral stationary phases has become the most popular and effective method used for the analytical and preparative separation of optically active compounds. This review mainly focuses on the recent development trends for novel chiral stationary phases based on chitosan derivatives, cyclofructan derivatives, and chiral porous materials that include metal‐organic frameworks and covalent organic frameworks in high‐performance liquid chromatography. The enantioseparation performance and chiral recognition mechanisms of these newly developed chiral selectors toward enantiomers are discussed in detail.     

Capillary gas chromatography separation of pyrethroic acid methyl esters using four acylated cyclodextrin derivatives as chiral stationary phases

Summary Four cyclodextrin derivatives (CDs) were synthesized by substituting 3-OH of 2,6-di-O-pentyl-β-cyclodextrin with four different chain lengths of acyl groups (butyryl, valeryl, heptanoyl, octanoyl). The chromatographic properties of the four CD derivatives as stationary phases of capillary gas chromatography (CGC) were investigated. These CDs exhibit a wide range of application. Not only five pairs of enantiomers of pyrethroic acid methyl esters were separated on the four CDs, but also some other racemic compounds. Among the four CDs, 2,6-di-O-pentyl-3-O-butyryl-β-CD possesses better enantiomer separation abilities to the studied enantiomers of pyrethroic acid methyl esters than the other studied CDs. The extension of chain length of the acyl groups in 3-position of CDs cannot improve the enantiomer separation abilities of the CD derivatives.     

Enantiomeric separation of amino alcohols by gas chromatography on a chiral stationary phase; influence of the perfluoroacylating reagent on the separation

Racemic amino alcohols have been separated as perfluoroacylated derivatives by gas chromatography using either improved Chirasil-Val or heptakis(2,6-di-O-methyl-3-O-pentyl)-β-cyclodextrin as stationary phase. Using Chirasil-Val all the amino alcohols investigated were separated to baseline (α values between 1.03 and 1.08) whereas only a few amino alcohols were resolved on the modified cyclodextrin column. The enantioselectivity obtained on the latter phase was, however, significantly higher. The separations were performed as trifluoroacetyl, pentafluoropropionyl, and heptafluorobutyryl derivatives and the chiral discrimination observed for the different derivatives was significantly different for both stationary phases. In oder to obtain a better understanding of the separation mechanism, the Gibbs-Helmholtz parameters Δ_(R,S)ΔH° and Δ_(R,S)ΔS° were determined. The most extraordinary result was obtained for the trifluoroacetyl derivative of allo-threoninol. In addition to the order of elution of the enantiomers being the opposite of that for the other compounds, the separation seems to be entropy controlled (the sign Δ_(R,S)ΔH° is positive), i.e. the separation improved at higher temperatures.     

Chromatographic Resolution of Racemates. New analytical methods (17)

A number of racemates can be resolved into optically pure enantiomers by chromatography on optically active adsorbents. Synthetic polymers with optically active amide, amino acid, and crown ether groups, natural products such as starch and cellulose, and also microcrystalline triacetylcellulose are suitable for this purpose. Racemates can also be resolved by gas chromatography on optically active stationary phases.     

Flow methods in chiral analysis

The methods used for the separation and analytical determination of individual isomers are based on interactions with substances exhibiting optical activity. The currently used methods for the analysis of optically active compounds are primarily high-performance separation methods, such as gas and liquid chromatography using chiral stationary phases or chiral selectors in the mobile phase, and highly efficient electromigration techniques, such as capillary electrophoresis using chiral selectors. Chemical sensors and biosensors may also be designed for the analysis of optically active compounds.     

Resolution of amino acids on optically active stationary phase by gasChromatography

Summary The various enantiomeric esters of TFA--amino acid may be separated directly by gas chromatography on optically active stationary phase. In this work we investigated the resolution of relatively volatile amino acids (alanine, -amino-n-butyric acid, valine, threonine, allo-isoleucine, isoleucine, norvaline, leucine, proline, norleucine, serine) and less volatile amino acids (cysteine, aspartic acid, methionine, phenylalanine, glutamic acid) on a glass capillary column coated with N-caproyl-L-valyl-L-valine cyclohexyl ester.     

Enantioselective capillary gas chromatography on the basis of host-guest interactions with modified cyclodextrins

The use of per-O-alkylated as well as partially alkylated/acylated derivatives of α-, β-, and γ-cyclodextrins as chiral stationary phases has opened many new applications to enantioselective gas chromatography. Chiral recognition is now independent of hydrogen bonding interactions and enantiomer separations are achieved for a large variety of compounds which could not be separated before, including lactones, spiroacetals, alkyl halides, olefins, and even compounds with axial asymmetry.     

2-Acyl-dimedones as UV-active protective agents for chiral amino acids: enantiomer separations of the derivatives on chiral anion exchangers

2-Acetyldimedone and 12 related compounds were employed as UV-active pre-column derivatizing agents for amino acids. Direct enantioseparation of the products was achieved using chiral anion exchanger stationary phases in polar-organic mobile phase mode. Under basic conditions, the reagents´ cyclic β-tricarbonyl motifs can give rise to exo- and endocyclic enols through tautomerization. However, with primary amines (proteinogenic and unusual amino acids, aminosulfonic and aminophosphonic acids), we exclusively observed the formation of exocyclic enamine-type products. Reaction yields depended strongly on the 2-acyl modification of the reagent; in particular, we observed a significant decrease when electronegative or sterically demanding substituents were present in α-position to the exocyclic carbonyl group. In addition to improving UV detectability of the products, the introduction of this protective group facilitated successful enantiomer separations of the amino acid derivatives on Cinchona-based chiral anion exchangers. Particularly high enantiomer selectivity was observed in combination with stationary phases bearing a new variation of selectors with π-acidic (electron-poor) bis(trifluoromethyl)phenyl groups. No racemization of the analytes occurred at any stage of the analytical method including the deprotection, which was achieved with hydrazine.

2,6-Di-O-pentyl-3-O-propionyl-γ-cyclodextrin as an enantiomeric stationary phase for capillary gas chromatography

Summary 2,6-Di-O-pentyl-3-O-propionyl--cyclodextrin has been synthesized and used for enantiomer separation in capillary gas chromatography. Experimental results showed that the stationary phase has good enantiomer selectivity toward amino acids, amines, alcohols, diols, epoxides, and lactones,etc. 2,6-Di-O-pentyl-3-O-propionyl--cyclodextrin has good thermal stability: the highest recommended temperature isca 170°C.     

Gas Chromatographic Separation of Enantiomers on Cyclodextrin Derivatives

In investigations concerned with the phenomenon of molecular chirality, the use of gas chromatography for the enantiomeric analysis of stable, volatile compounds is a technique of steadily growing importance. ^[1] In the last three years an important breakthrough in gas-chro-matographic separation of enantiomers has been achieved by using alkylated cyclodextrins (α, β, and γ) as chiral stationary phases in high-resolution capillary columns. In academic and commercial practice two different and complementary strategies have been adopted up to now. In the first, alkylated cyclodextrins are diluted with polysiloxanes and coated on glass or fused silica capillary columns. In the second, lipophilic per-n-pentylcyclodextrins and hydrophilic di-n-pentyl- and hydroxyalkylpermethylcyclodextrins are coated directly in the form of liquid phases onto suitably pretreated glass or fused silica surfaces. These techniques permit enantiomer separations not only for polar diols and alcohols, derivatized hydroxycarboxylic acids, amino acids, sugars, and alkyl halides, but also for nonpolar alkenes, cyclic saturated hydrocarbons, and metal π complexes. An important aspect for practical applications is that in many cases the enantiomers can be separated without previous derivatization. Whereas the resolution of racemates of unfunctionalized hydrocarbons is attributed to an enantioselective host–guest inclusion complex, some observations indicate that for polar guest molecules additional enantioselective interactions are also involved. The new chiral stationary phases can be used over a wide range of temperatures (25 to 250°C). The technique described is likely to become widely adopted as a simple, accurate and highly sensitive method for the enantiomeric analysis of chiral compounds that can be vaporized without decomposition. It will also stimulate future research aimed at finding universal cyclodextrin phases and elucidating the mechanisms of enantioselectivity.     

同时烙印分子烙印手性固定相

对采用两种对本同时烙印的方法,制备的氨基酸衍生物分子烙印手性固定相进行了考察。研究表明,如果两种烙印分子单独烙印的分子烙印手性对固定相对两种烙印分子具有较强的手性交叉拆分能力,那么这两种烙印分子同时烙印制得的分子烙印手性固定相就可对两种烙印分子均具有很好的手性分离能力,从而使专一性强的分子烙印手性固定相能同时分离多种对映体。     

新型高温固定相C60聚硅氧烷的合成及色谱特性

合成了两种新的C₆₀侧链线性聚硅氧烷,并用作毛细管气相色谱固定相.该类固定相柱效高,使用温度范围宽,热稳定性好,最高柱温达360℃,基线漂移量为3×10^-14～4×10^-14nm,对各类位置异构体有独特的分离效果,尤其对高沸点化合物如糖、多环芳烃和邻苯二甲酸酯类有良好的分离选择性.     

Cyclodextrins as chiral stationary phases in capillary gas chromatography. Part V: Octakis(3-O-butyryl-2,6-di-O-pentyl)-γ-cyclodextrin

γ-Cyclodextrin with 3-O-butyryl and 2,6-di-O-pentyl residues is a very versatile chiral stationary phase for enantiomer separation. Most of the common and many uncommon amino acids can be separated as well as α- and β-hydroxy acids, chiral alcohols, diols, triols, ketones, bicyclic, and tricyclic acetals, amines, alkyl halides, lactones, and functionalized cyclopropane derivatives.