Characterization of cyclofructan-based chiral stationary phases by linear free energy relationship

Cyclofructans (CFs), a new class of chiral selectors, have been recently introduced for application in liquid chromatography and capillary electrophoresis. So far, derivatized CFs have performed interesting separation possibilities for a variety of compounds. The current work is focused on characterization of three different CF-based chiral stationary phases (CF-based CSPs), i.e. isopropyl carbamate cyclofructan 6 (IP-CF6), R-naphthylethyl carbamate cyclofructan 6 (RN-CF6) and dimethylphenyl carbamate cyclofructan 7 (DMP-CF7). The linear free energy relationship (LFER) model was used to reveal the dominant interactions participating in the complex retention mechanism. A set of 44 different test solutes, with known solvation parameters, was used to determine the regression coefficients of the LFER equation under two mobile-phase compositions in normal separation mode. The LFER results showed that hydrogen bond acidity, hydrophobicity and dipolarity/polarizibility mostly affect the retention and separation process on the CF-based columns in the studied separation systems.     

Properties of two amide‐based hydrophilic interaction liquid chromatography columns

Hydrophilic interaction liquid chromatography is a separation technique suitable for the separation of moderately and highly polar compounds. Various stationary phases (SPs) for hydrophilic interaction liquid chromatography are commercially available. While the SPs based on the same type of ligand are available from different providers, they can display a distinct retention characteristics and separation selectivity. The current work is focused on characterization and comparison of the separation systems of two amide‐based HPLC columns from two producers, i.e. XBridge Amide column and TSK gel Amide‐80 column. Several characterization procedures (tests) were used to investigate the differences between these columns. The chromatographic behavior of selected analytes indicates that multimodal interactions are responsible for retention and separation on these columns. Multiple testing approaches were used in order to reveal subtle differences between the SPs. Both amide‐based columns showed certain differences in retention, selectivity, and plate counts. Based on the tests used in this study, we conclude that the investigated columns provide a different degree of H‐bonding interactions.     

梯度加压毛细管电色谱同时分离大黄提取液中5种蒽醌类化合物

建立了同时分离药用大黄提取液中大黄酸、芦荟大黄素、大黄素、大黄酚和大黄素甲醚5种蒽醌类活性成分的梯度加压毛细管电色谱的新方法.实验结果显示,大黄提取液中的5种蒽醌化合物可在22min内完全分离,梯度洗脱微柱液相色谱的柱效为等度洗脱微柱液相色谱的6.63倍,梯度毛细管电色谱的柱效为梯度微柱液相色谱的4.6倍.     

Two-dimensional high performance liquid chromatography for the separation of complex mixtures of explosives and their by-products

Summary The insufficient ability of one dimensional HPLC to separate complex mixtures such as environmental samples can be overcome by using two dimensional systems combining columns with alternative/orthogonal selectivities. Such a system for the separation of complex mixtures of explosives, their by-products and degradation products from environmental samples was developed and evaluated. It makes use of the different retention characteristics of an alkyl modified silica and a safrol modified silica in the reversed phase mode. The high peak capacity of two dimensional systems predicted by theory was realized employing a flexible switching technique and utilizing differences in the elution strength of the mobile phases. Thus, peak compression on the head of the second column was exploited. The efficiency of the two dimensional system was demonstrated for the separation of a complex mixture of nitroaromatic reference compounds. Furthermore, the system was applied to separate and identify nitro and nitroamino organic compounds in a groundwater sample from a former ammunition plant. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok Hungary, September 3–5, 1997     

Hydrophilic interaction chromatography

Separation of polar compounds on polar stationary phases with partly aqueous eluents is by no means a new separation mode in LC. The first HPLC applications were published more than 30 years ago, and were for a long time mostly confined to carbohydrate analysis. In the early 1990s new phases started to emerge, and the practice was given a name, hydrophilic interaction chromatography (HILIC). Although the use of this separation mode has been relatively limited, we have seen a sudden increase in popularity over the last few years, promoted by the need to analyze polar compounds in increasingly complex mixtures. Another reason for the increase in popularity is the widespread use of MS coupled to LC. The partly aqueous eluents high in ACN with a limited need of adding salt is almost ideal for ESI. The applications now encompass most categories of polar compounds, charged as well as uncharged, although HILIC is particularly well suited for solutes lacking charge where coulombic interactions cannot be used to mediate retention. The review attempts to summarize the ongoing discussion on the separation mechanism and gives an overview of the stationary phases used and the applications addressed with this separation mode in LC.     

Ionic liquid-modified silica as a new stationary phase for chromatographic separation

A new stationary phase based on silica modified with 1-methyl-3-propylimidazolium chloride was synthesized and characterized in this paper. A derivative of 1-methyl-3-propylimidazolium chloride was used to chemically modify the surface of silica particles to act as the stationary phase for HPLC. The modified particles were characterized by Fourier Transform Infrared (FT-IR), ¹³C NMR spectroscopy and thermogravimetric analysis (TGA). The surface modification procedure rendered particles with a surface coverage of 0.89 μmol/m² of alkylimidazolium chloride. Columns packed with the modified silica and blank silica particles were tested under HPLC conditions. Preliminary evaluation of the stationary phase for HPLC was performed using aromatic compounds as model compounds. The separation mechanism appears to involve multiple interactions including ion exchange, hydrophobic and electrostatic interactions.     

Isolation,Separation, and Preconcentration of Biologically Active Compounds from Plant Matrices by Extraction Techniques

Development of efficient methods for isolation and separation of biologically active compounds remains an important challenge for researchers. Designing systems such as organomineral composite materials that allow extraction of a wide range of biologically active compounds, acting as broad-utility solid-phase extraction agents, remains an important and necessary task. Selective sorbents can be easily used for highly selective and reliable extraction of specific components present in complex matrices. Herein, state-of-the-art approaches for selective isolation, preconcentration, and separation of biologically active compounds from a range of matrices are discussed. Primary focus is given to novel extraction methods for some biologically active compounds including cyclic polyols, flavonoids, and oligosaccharides from plants. In addition, application of silica-, carbon-, and polymer-based solid-phase extraction adsorbents and membrane extraction for selective separation of these compounds is discussed. Potential separation process interactions are recommended; their understanding is of utmost importance for the creation of optimal conditions to extract biologically active compounds including those with estrogenic properties.     

Sequential fractionation procedure for the identification of potentially cytochrome P4501A-inducing compounds

A multistep fractionation procedure for the separation of nonpolar aromatic compounds with respect to cytochrome P4501A induction is presented. Normal-phase HPLC on nitrophenylpropyl silica and cyanopropyl silica was tested for group-specific separation as a first fractionation step. Subsequent individual compound-specific PAH fractionation was done by means of reversed-phase HPLC. Electron-donor-acceptor HPLC and size-exclusion chromatography were applied to separate PAHs, PCBs, PCNs and PCDD/Fs according to their number of aromatic carbon atoms, their hydrophobicity, their degree of chlorination, their planarity and their molecular size. The method was validated for complex environmental mixtures on the basis of two sediment extracts.     

Molecular Modeling of Cyclodextrin-Guest Molecule Interactions

Abstract

Inclusion complexes formed with substituted aromatic compounds and ß-cyclodextrin were studied using molecular modeling techniques. Energies of the MM2 optimized structures were correlated with HPLC retention times. A direct correlation between the inclusion complex stability and the HPLC retention times for the bonded ß-cyclodextrin was found within a series of disubstituted aromatic compounds. Evidence for a secondary mechanism of separation was found.     

Purification method for the isolation of monophosphate nucleotides from Champagne wine and their identification by mass spectrometry

Monophosphate nucleotides are difficult to identify in Champagne wine because they are present in small concentrations in a complex mixture. A method for the isolation, separation and identification of reference compounds, which achieved on average 79% recovery (except for cytidine derivatives), was developed and applied to wine. Some monophosphate nucleotides were then isolated from a Champagne wine aged on lees for 8 years, by ultrafiltration followed by a semi-preparative HPLC step using a strong anion-exchange column. The fraction obtained was subjected to HPLC in a reversed-phase column to remove the salt previously introduced, before identification of compounds by HPLC coupled to a mass spectrometer. For the first time in wine, 5'-IMP, 5'-AMP, 5'-CMP, 5'-GMP, 5'-UMP and the 3'- and/or 2'-isomers of the four latter compounds were identified by comparing their HPLC and electrospray ionization mass spectrometry data with those of reference nucleotides.     

Two-dimensional reversed-phase liquid chromatography using two monolithic silica C18 columns and different mobile phase modifiers in the two dimensions

Comprehensive two-dimensional (2D) HPLC in the reversed-phase liquid chromatography (RPLC) mode using C18 silica monolith columns at first dimension (1st-D) (10 cm x 4.6mm I.D.) and second dimension (2nd-D) (5 cm x 4.6mm I.D.) was carried out successfully. A mixture of water and tetrahydrofuran (THF) was used as a mobile phase in the 1st-D separation, and a mixture of water and methanol (CH3OH) in the 2nd-D separation. Sample fractions from 1st-D column were directly loaded into an injection loop of the 2nd-D HPLC equipped with two injector valves for one column. The fractionation time at the 1st-D that was equal to the separation time at the 2nd-D was 45 or 60s. Total peak capacity up to 900 was obtained in about 60 min for the isocratic mode separation of aromatic compounds in this system. Gradient elution mode applied to both 1st-D and 2nd-D separations resulted in shorter separation time and better separation efficiencies than the isocratic mode. It was demonstrated that 2D-HPLC systems employing popular C18 stationary phases with different organic modifiers in mobile phases for each dimension could produce large peak capacity. The different selectivities were provided by the difference in polar interactions between a solute and the organic modifier existing in the stationary phase.     

Benefits and Pitfalls of HPLC Coupled to Diode-Array,Charged Aerosol,and Coulometric Detections: Effect of Detection on Screening of Bioactive Compounds in Apples

The new screening method for rapid evaluation of major phenolic compounds in apples has been developed. Suitability of coupling HPLC/UHPLC separation with the diode-array detection and universal charged aerosol detection with respect to the presence of interfering substances was tested. Characteristics of both detection techniques were compared and method linearity, limits of detection and quantitation, and selectivity of them determined. Student t-test based on slopes of calibration plots was applied for the detailed comparison. The diode-array detection provided the best results regarding sensitivity and selectivity of the developed method in terms of evaluation of phenolics profiles. The response of the charged aerosol detector was negatively affected by co-eluting substances during rapid-screening analyses. Coulometric detection was used for advanced characterization of extracts in terms of antioxidant content and strength to obtain more complex information concerning sample composition. This detection also allowed evaluation of unidentified compounds with antioxidant activity. HPLC/UHPLC separation using a combination of diode-array and coulometric detectors thus represented the best approach enabling quick, yet complex characterization of bioactive compounds in apples.     

Impact of methanol and acetonitrile on separations based on pi-pi interactions with a reversed-phase phenyl column

Studies were performed to investigate the roles of methanol and acetonitrile on the retention mechanism of an active pharmaceutical ingredient (API) and related compounds with a reversed phase phenyl column. Different retention orders were observed depending upon whether acetonitrile or methanol was used as the organic modifier. We propose that acetonitrile impedes the selective pi-pi interactions between the analyte molecules and the phenyl groups in the stationary phase. Further study with 1-naphthoic acid and 1-naphthol as test compounds in the HPLC separation provides additional support for the influence of acetonitrile on pi-pi interactions between analyte molecules and a phenyl stationary phase. This study suggests that methanol be used as the preferred organic modifier with phenyl columns to achieve selectivity based upon pi-pi interactions.     

Comparative study on the enantiomer separation of 1,1'-binaphthyl-2,2'diyl hydrogenphosphate and 1,1'-bi-2-naphthol by liquid chromatography and capillary electrophoresis using single and combined chiral selector systems

The chiral recognition ability of single and dual selectors, that were used as additives, have been investigated by HPLC and CE. Native beta- and gamma-cyclodextrins, permethylated beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, cholic acid and taurodeoxycholic acid sodium salts were applied as chiral selectors, whereas the atropisomers of 1,1'-binaphthyl-2,2'-diyl hydrogenphosphate, and 1,1'-bi-2-naphthol served as model compounds. It was found that all investigated selectors, except for gamma-cyclodextrin, display the same affinity pattern for binaphthyl enantiomers, i.e., binding the S more strongly than the R enantiomer. However, the differences in the phase distribution of chiral selectors led to the opposit elution order of enantiomers: with cyclodextrins, the first eluted is S enantiomer, while R is the first eluted for bile salts. Under the conditions studied, cyclodextrins (except gamma-cyclodextrin), as well as cholic acid sodium salts acting singly, enable the separation of 1,1'-binaphthyl-2,2'-diyl hydrogenphosphate enantiomers both by HPLC and CE methods, while 1,1'-bi-2-naphthol enantiomers were resolved only under CE conditions with permethylated cyclodextrin or bile salts. In both techniques the application of dual systems could improve resolution or make it worse (oreven cancel), depending on the sign of enantioselectivity of particular selectors, their concentrations and localization: mobile or stationary phase. It has been found that the mechanism of separation as well as interactions occurring between two selectors may be followed by using combined HPLC and CE methods. The obtained results proved that, as well as beta-CD, TM-beta-D and gamma-CD also form inclusion complexes with cholic acid sodium salts. The reversal of elution order may be realized by two procedures: changing a single selector, i.e., cyclodextrin on cholic acid sodium salt or vice versa, and by changing the proportion of selectors in the combined bile salt-cyclodextrin system.     

A simple model describing the retention behavior of octreotide and its glycosylated derivatives in reversed phase HPLC

Summary The retention behavior of octreotide, a somatostatin analogue, and its glycosylated derivatives containing different numbers of glucose units has been studied by reversed phase HPLC. A retention model was developed by correlating the logarithm of the retention factor with the hydrophilic-lipophilic balance of the analyte. Linear functions could be derived for all the separation systems investigated. The slopes of the straight lines were a measure of the selectivity of the chromatographic system and enabled calculation of increments for the saccharide groups in different eluent systems. The highest increment was found using trifluoroacetic acid (TFA) as ion pairing agent. The model was extended to substitution of the same peptide with hydrophobic groups such as acetyl and alkyl. Straight lines were again obtained. The influence of the different eluent systems upon peak shape and retention is also discussed. Owing to the strong peak tailing a dual retention mechanism consisting of hydrophobic and silanophilic interactions was assumed. It was shown that addition of quaternary ammonium compounds to mask the surface silanols of the stationary phase reduced both the peak tailing and the retention of the peptides.     

Selective detection of metal species in HPLC and FIA by means of pulsed amperometric detection (PAD)

Triple-step pulsed amperometry is used to detect selectively metal complex species after HPLC separation in the presence of coeluting matrix compounds. Optimized pulse parameters enable accurate and selective detection of species even in the presence of electroactive compounds such as phenols or sulfurcontaining peptides. Complexes of tin(IV) with rutin and of platinum(II) with glutathione are presented as examples for these classes of compounds. It is demonstrated that by changing the pulse waveform direct and indirect detection modes can be realized. The method is applied to detect platinum species in a grass extract and iron(II)-lactate in fruit juice. Furthermore it is shown, that pulsed amperometry can be used as a detector in FIA for determining the fraction of metal complex formed after mixing of metal and excess ligand without separation.     

Comparison of separation power of ultra performance liquid chromatography and comprehensive two-dimensional liquid chromatography in the separation of phenolic compounds in beverages

In this study, 1-D and 2-D liquid chromatographic systems, namely, conventional HPLC, UPLC, HPLC x HPLC and HPLC x UPLC systems were developed and evaluated for the separation of phenolic acids in wine and juices. In the LC x LC studies, the first dimension separation was based on RPLC and the second dimension was performed with ion-pair chromatography. Three different columns, namely two short columns packed with either 2.5 or 1.7 microm particles and a monolithic column, were tested for the fast second dimension separation. The best results were obtained when the monolithic column was applied for the second dimension separation. The peak capacities for comprehensive 2-D systems varied from 330 to 616.     

对映异构体的高效毛细管电泳分离与测定

高效毛细管电泳是８０年代发展起来的一种高效、快速的新型分离技术，在对映异构体分离方面有着广泛的应用前景，本文介绍了这一新型分离技术用于对映体分离的基本原理，并列举了一些对映异构体分离的实例。