Trennung und Charakterisierung von β-Carotin-Isomeren

Separation and Characterization of the cis-Isomers of β,β-Carotene A stable HPLC. system is described allowing the excellent separation of 11 different cis-isomers of β,β-carotene from the all-trans compound. The system is applied to the analysis of cis/trans mixtures obtained from plant extracts and by photoisomerization of the all-trans isomer. Al₂O₃ is used as the stationary phase while hexane with controlled H₂O content is utilized as the mobile phase. With the aid of the optimum conditions 8 sufficiently stable cis isomers were isolated and their structures shown to be the 9-, 13- and 15-cis, the 9,9′-, 9, 13-, 9, 13′- and 13,13′-di-cis and, tentatively, the 9,13,13′-tri-cis β,β-carotenes by application of 270-MHz-FT.-¹H-NMR. spectroscopy.     

Capillary GC using pyridyl β‐cyclodextrin stationary phase

A new β‐CD derivative, heptakis [2,6‐di‐O‐pentyl‐3‐O‐(4′‐chloro‐5′‐pyridylmethyl)]‐β‐CD, was synthesized by the selective introduction of a pyridyl group on the 3‐positions of β‐CD. The chromatographic properties of the pyridyl β‐CD derivative were studied by using it as the stationary phase in capillary GC. The polarity of the prepared stationary phase was moderate, and the separation results demonstrated that the prepared stationary phase possessed excellent separation ability and chiral recognition for a wide range of analytes. Not only the aromatic positional isomers, such as o‐, m‐, p‐xylene and α‐, β‐naphthol isomers, but also some compounds with multi‐stereogenic centers, such as n‐(1‐methylpropyl)‐3‐(2,2‐dichloroethenyl)‐2,2‐dimethylcyclopropanecarboxamide and n‐(1‐methylpropyl)‐3‐(2‐chloro‐3,3,3‐trifluoropropenyl)‐2,2‐dimethylcyclopropanecarboxamide with three stereogenic centers including eight configurational isomers, were successfully separated. The results also indicated that the polarity of the β‐CD derivative, and the hydrogen bonding between the β‐CD derivative, and the analytes had a very important effect on separation.     

Use of Chemically Bonded β‐Cyclodextrin Silica Stationary Phase for Liquid Chromatographic Separation of Structural Isomers

The retention behavior of five disubstituted benzene derivatives and two naphthalene derivatives is examined by using a chemically bonded β‐cyclodextrin silica stationary phase with the moiety containing the s‐triazine. The chromatographic results of five disubstituted benzene derivatives and two naphthalene derivatives show that effective separation is achieved on this stationary phase by high‐performance liquid chromatography. The results of the present investigation indicate that the formation of inclusion complexes plays a dominant role in the separation mechanism. However, the selectivity can be significantly enhanced by the n‐n interactions between the s‐triazine ring of the chemically bonded β‐cyclodextrin silica stationary phase and the aromatic ring of solutes. For example, the effective separation of the o‐, m‐, and p‐toluidine isomers on this stationary phase with the moiety containing the s‐triazine ring was better than on that of some β‐cyclodextrin bonded stationary phases without the moiety containing s‐triazine ring.     

Synthesis and chromatographic evaluation of phenyl/tetrazole bonded stationary phase based on thiol‐epoxy ring opening reaction

A silica‐based reversed‐phase stationary phase bonding with phenyl and tetrazole groups was synthesized by thiol‐epoxy ring opening reaction. The bonded groups could not only provide hydrophobic interaction, but also π–π, hydrogen bonding, electrostatic interactions, and so on. The results of characterization with elemental analysis and solid‐state ¹³C cross‐polarization magic‐angle‐spinning NMR spectroscopy indicated the successful preparation of phenyl/tetrazole sulfoether bonded stationary phase. Chromatographic evaluation revealed that phenyl/tetrazole sulfoether bonded stationary phase behaved well under the reversed‐phase mode. The column parameters (H, S*, A, B, and C) showed different selectivity compared with some typical commercial columns, and it was validated by the separation of estrogen, ginsenoside, alkaloid samples. Based on the different selectivity between phenyl/tetrazole sulfoether bonded stationary phase and C18 columns, phenyl/tetrazole sulfoether bonded stationary phase also showed potential to construct a 2D reversed‐phase liquid chromatography system with C18. And it was verified by the separation of corydalis tuber and curcuma zedoary extracts.     

Development and application of a new 25,27‐bis(l‐phenylalaninemethylester‐N‐carbonylmethoxy)‐26,28‐dihydroxy‐para‐tert‐butylcalix[4]arene stationary phase

A 25,27‐bis(l ‐phenylalaninemethylester‐N‐carbonylmethoxy)‐26,28‐dihydroxy‐ para‐tert‐butylcalix[4]arene‐bonded silica gel stationary phase was synthesized, structurally characterized and used for LC. Its separation mechanism was studied and compared with octadecyl‐bonded stationary phase, as well as our previously prepared para‐tert‐butylcalix[4]arene‐1,2‐crown‐4 stationary phase. Meanwhile, the chromatographic behaviors were investigated by using polycyclic aromatic hydrocarbons, monosubstituted benzenes, anilines, phenols, Tanaka tests solutes, fluoroquinolones, and flavonoids as probes. Mechanisms involved in the chromatographic separation included hydrophobic, π‐π and π‐electron transfer, hydrogen bonding, and inclusion interactions. Moreover, the column was successfully employed for the analysis of the illegal additive of melamine in milk product.     

Effect of analyte lipophilicity on the resolution of α‐ and β‐amino acids on liquid chromatographic ligand exchange chiral stationary phases

Separation of the two enantiomers of racemic α‐ and β‐amino acids on two ligand exchange chiral stationary phases (CSPs) prepared previously by covalently bonding sodium N‐((S)‐1‐hydroxymethy‐3‐methylbutyl)‐N‐undecylaminoacetate or sodium N‐((R)‐2‐hydroxy‐1‐phenylethyl)‐N‐undecylaminoacetate on silica gel was studied with variation of the organic modifier (methanol) concentration in the aqueous mobile phase. In particular, the variation of retention factors with changing organic modifier concentration in the aqueous mobile phase was found to be strongly dependent on both the analyte lipophilicity and the stationary phase lipophilicity. In general, the retention factors of relatively lipophilic analytes on relatively lipophilic CSPs tend to increase with increasing organic modifier concentration in the aqueous mobile phases while those of less lipophilic or hydrophilic analytes tend to increase. However, only highly lipophilic analytes show decreasing retention factors with increasing organic modifier concentration in the aqueous mobile phase on less lipophilic CSPs. The contrasting retention behaviors on the two CSPs were rationalized by the balance of the two competing interactions, viz. hydrophilic interaction of analytes with polar aqueous mobile phase and the lipophilic interaction of analytes with the stationary phase.     

Chromatographic behavior of a new hybrid type RP material containing silica bonded 1,3‐alternate 25,27‐bis‐[cyanopropyloxy]‐26,28‐bis‐[3‐propyloxy]‐calix[4]arene

A novel 1,3‐alternate 25,27‐bis‐[cyanopropyloxy]‐26,28‐bis‐[3‐propyloxy]‐calix[4]arene‐bonded silica gel stationary phase (CalixPrCN) was prepared and its structure was confirmed by ATR‐FTIR spectroscopy and elemental analysis. The CalixPrCN phase was characterized in terms of its surface coverage, hydrophobic selectivity, aromatic selectivity, shape selectivity, hydrogen bonding capacity, residue metal content, and silanol activity based on Tanaka, Lindner, and SMR 870 test protocols. The effect of the acetonitrile content on the retention and selectivity of the selected neutral, basic, and acidic solutes was studied. The neutral and acidic analytes exhibited classical RP behavior, in which retention time decreases with increasing acetonitrile content. In contrast, basic analytes showed an increase in retention at low and high percentages of acetonitrile, forming “U‐shaped” retention profiles. The new calixarene phase was compared with previously reported 1,3‐alternate 25,27‐bis‐[propyloxy]‐26,28‐bis‐[3‐propyloxy]‐calix[4]arene stationary phase and commercial cyanopropyl column. The results indicate that the CalixPrCN stationary phase behaves like RP packing; however, inclusion complex formation, dipole–dipole, and π–π interactions seem to be involved in the separation process. The selectivity of this phase was demonstrated in separation of polynuclear aromatic hydrocarbons, non‐steroidal anti‐inflammatory drugs, and sulfonamides as analytes.     

Preparation and evaluation of a new synthetic polymeric chiral stationary phase for HPLC based on the trans-9,10-dihydro-9,10-ethanoanthracene-(11S,12S)-11,12-dicarboxylic acid bis-4-vinylphenylamide monomer

A new synthetic polymeric chiral stationary phase for liquid chromatography was prepared via free-radical-initiated polymerization of trans-9,10-dihydro-9,10-ethanoanthracene-(11S,12S)-11,12-dicarboxylic acid bis-4-vinylphenylamide. The new polymeric chiral stationary phase (CSP) showed enantioselectivity for many chiral compounds in multiple mobile phases. High stability and sample capacities were observed on this polymeric chiral stationary phase. Mobile phase components and additives affected chiral separation greatly. This new synthetic chiral stationary phase is complementary to two other related commercially available CSPs: the P-CAP and P-CAP-DP columns. Interactions between the chiral stationary phase and analytes that lead to retention and chiral recognition include hydrogen bonding, dipolar, and π–π interactions. Repulsive (steric) interactions also contribute to chiral recognition. Figure LC chromatograms showing the analytical (blue) and preparative (red) separations of N-(3,5-dinitrobenzoylleucine) enantiomers on a new synthetic polymeric chiral stationary phase     

Effect of pH and mobile phase additives on the chromatographic behaviour of an amide‐embedded stationary phase: Cyanocobalamin and its diaminemonochloro‐platinum(II) conjugate as a case study

Several mobile phase additives (i.e., organic acids and their ammonium salts) were used to modulate the chromatographic retention of cyanocobalamin and its cis‐diaminemonochloroplatinum(II) conjugate, depending on the specific nature of the stationary phase. Regardless of the mobile phase additive, the positively charged cyanocobalamin‐cis‐diaminemonochloroplatinum(II) conjugate was systematically less retained than cyanocobalamin on a conventional octadecyl‐silica column. In contrast, the amide‐embedded C18 column exhibited a progressive increase in the conjugate retention time upon changing the mobile phase additive from organic (acetic, formic and trifluoroacetic) acids to ammonium salts, ultimately leading to an inversion of the elution order. This change of retention was interpreted by invoking the interplay between hydrophobic interactions, hydrogen bonding between the conjugate and the polar amide groups and the ion‐pairing ability of the lyophilic counterions, whereby the acetate anion was found to be the most suitable to control the solute retention.     

Effect of Anchimeric Assistance in the Reaction of Triphenylphosphine with α,β‐Unsaturated Carboxylic Acids

Quaternization of triphenylphosphine with maleic and cis‐aconitic acids is strongly accelerated by participation of the cis‐carboxyl group in stabilization of the phosphonium zwitterion intermediate by intramolecular hydrogen bonding, in spite of steric hindrance by the acid's reaction center. A similar effect for trans‐isomeric acids is not observed, which can be rationalized on the basis of spatial structures of the generated zwitterions, implying an electrostatic interaction between the phosphonium center and carbonyl oxygen atom. The effect of anchimeric assistance decreases when the intramolecular hydrogen bonding disfavors attack of the phosphine on the sterically less hindered carbon atom of the C=C bond, as observed for cis‐aconitic acid.     

Phase Transition in Trans and Cis-1,2-Cyclohexanediol Studied by Infrared Spectroscopy

The study of trans- and cis-1,2-cyclohexanediol by infrared spectroscopy was performed. The variation of the maximum frequency and of the bandwidth of the OH stretching vibration give evidence of the role played by hydrogen bonding in the solid and liquid phases of both isomers and allows to follow the phase transitions. A solid rotator phase is shown for the cis compound.This revised version was published online in November 2005 with corrections to the Cover Date.     

Preparation and characterization of phenyl carbamate derivative β-cyclodextrin bonded phase for chiral HPLC

A partially substituted β-cyclodextrin chiral stationary phase was prepared by the reaction of phenyl isocyanate. The enantiomers of a series of O,O-diethyl(p-methylbenzenesulfonamido)-aryl(or alkyl)-methylphosphonates were studied on the prepared phenyl carbamate derivative β-cyclodextrin bonded phase and a commercial (S)-(+)-1–(1-naphthyl) ethylcarbamate derivative β-cyclodextrin bonded phase on normal phase chromatographic condition. Results show that the prepared phenyl carbamate derivative β-cyclodextrin bonded phase has better enantiomeric selectivity to the series of compounds. A chiral recognition mechanism was suggested for the separation of these novel organic phosphorus enantiomers.     

Preparation,characterization and application of a new 25,27‐bis‐[2‐(5‐methylthiadiazole)thioethoxyl]‐26,28‐dihydroxy‐para‐tert‐butyl calix[4]arene stationary phase for HPLC

A new para‐tert‐butylcalix[4]arene column containing thiadiazole functional groups was prepared and used for the separation of polycyclic aromatic hydrocarbons, phenolic compounds, aromatic amines, benzoic acid and its derivatives by high‐performance liquid chromatography (HPLC). The effect of organic modifier content in the mobile phase on retention and selectivity of these compounds were investigated. The results indicate that the stationary phase behaves like reversed‐phase packing. However, hydrogen bonding, π–π and inclusion interactions seem to be involved in the separation process. The column has been successfully employed for the analysis of clenbuterol in pork and pig casing; the limit of detection and the limit of quantitation for this method by HPLC‐UV detection was 0.03 and 0.097 μg/mL, respectively; the method is demonstrated to be suitable and a competitive alternative analytical method for the determination of clenbuterol.     

Chromatographische Trennung und quantitative Bestimmung aller acht Stereoisomeren von α-Tocopherol

Separation and Quantitation of All Eight Stereoisomers of α-Tocopherol by Chromatography The results of the analysis of several derivatives of (all-rac)-α-tocopherol by HPLC on a chiral stationary phase and by capillary GC on a achiral stationary phase are reported. Consecutive application of both methods to the ethyl-ether derivative allows the separation and quantification of all eight possible stereoisomers of (all-rac)-α-tocopherol.     

Development of a decaaza‐cyclophane stationary phase for high‐performance liquid chromatography

A new stationary phase for high‐performance liquid chromatography was prepared by covalently bonding a heteroatom‐bridged cyclophane onto silica gel using 3‐aminopropyltriethoxysilane as the coupling reagent. The structure of the new material was characterized by infrared spectroscopy, elemental analysis, and thermogravimetric analysis. The linear solvation energy relationship method was successfully employed to evaluate the new phase with a set of 25 solutes, and compared with octadecylsilyl and p‐tert‐butyl‐calix[4]arene bonded stationary phases. The retention characteristics of the new phase are similar to the octadecylsilyl and conventional calixarene phases, and it also has distinctive features. In addition, the chromatographic behavior of the phase was illustrated by eluting alkylbenzenes and inorganic anions in the reversed‐phase mode and anion‐exchange mode, respectively. Thus, multi‐interaction mechanisms and mixed‐mode separation of the new phase can very likely guarantee its promising application in the analysis of complex samples. The column has been successfully employed for the analysis of triazines in milk, and it is demonstrated to be a competitive alternative analytical method for the determination of triazine herbicide residues.     

Application of bromoacetate‐substituted β‐CD‐bonded silica particles as chiral stationary phase for HPLC

Bromoacetate‐substituted [3‐(2‐O‐β‐cyclodextrin)‐2‐hydroxypropoxy]propylsilyl‐appended silica particles (BACD‐HPS), an important and useful synthetic intermediate for preparation of novel types of macrocycles‐capped β‐CD‐bonded silica particles including crown ether/cyclam/calix[4]arene‐capped β‐CD‐bonded silica particles, have been prepared and used as chiral stationary phase for HPLC. This synthetic stationary phase is characterized by means of elemental analysis. For the first time, the chromatographic behavior of BACD‐HPS was systematically evaluated with several disubstituted benzenes and some chiral drug compounds under both normal and RP conditions in HPLC. The results show that BACD‐HPS has excellent selectivity for the separation of aromatic positional isomers and chiral isomers of some drug compounds when used as stationary phase in HPLC.     

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.     

High-Performance liquid chromatographic separation of enantiomers of unusual amino acids possessing cycloalkane or cycloalkene skeletons on a chiral crown ether containing stationary phase

Summary Direct reversed-phase high-performance liquid chromatographic methods were developed for the separation and identification of the enantiomers of mono- and bicyclic racemic β-amino acids:cis- andtrans-2-aminocyclopentane-1-carboxylic acids,cis- andtrans-2-aminocyclohexane-1-carboxylic acids,cis- andtrans-2-amino-4-cyclohexene-1-carboxylic acids,diendo- anddiexo-3-aminobicyclo[2.2.1]heptane-2-carboxylic acids anddiendo- anddiexo-3-amino-5-bicyclo[2.2.1]heptene-2-carboxylic acids. Enantioseparation was carried out by the application of a chiral stationary phase, Crownpak CR(+). The conditions of separation were optimized by changing the temperature, the flow rate and the pH of the mobile phase. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997.     

A Comparison of the Retention Behaviour of Vitamin A and Some Metabolites Eluted from Four Different Reversed Phase High Performance Liquid Chromatographic Stationary Phases

Five retinoids, 13-cis-retinoic acid, 9-cis-retinoic acid, all-trans-retinoic acid, all-trans-retinol and 13-cis-retinal were isocratically separated from four different reversed phase high performance liquid chromatographic stationary phases. By taking advantage of the different retention mechanisms, present between the stationary phases and the analytes, the retinoids were separated with different elution orders using the same mobile phase composition. Two of the stationary phases appeared to have more possibilities to interact with the analytes than the usual hydrophobic interactions. The stationary phase with embedded polar groups showed hydrogen bonding properties and the calix[4]arene based stationary phase showed possibilities to form inclusion complexes with the analytes. These additional interactions appeared to benefit the separations of the analytes. This publication shows the benefits by isocratically separate retinoids employing other stationary phases than the conventional C₁₈ stationary phase.     

HPLC enantioseparation of β2‐homoamino acids using crown ether‐based chiral stationary phase

RP high‐performance liquid chromatographic methods were developed for the enantioseparation of eleven unusual β²‐homoamino acids. The underivatized analytes were separated on a chiral stationary phase containing (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid as chiral selector. The effects of organic (alcoholic) and acidic modifiers, the mobile phase composition and temperature on the separation were investigated. The structures of the substituents in the α‐position of the analytes substantially influenced the retention and resolution. The elution sequence was determined in some cases: the S enantiomers eluted before the R enantiomers.