Organic Modifier In Mobile Phase Research Articles

Preparation and enantioseparation characteristics of three chiral stationary phases based on modified β-cyclodextrin for liquid chromatography

In order to study the effect of the nature and the length of the spacer, three mixed 10-undecenoate/phenylcarbamate derivatives of beta-cyclodextrin have been prepared and linked to allylsilica gel by means of a radical reaction. The chiral recognition ability of the resulting materials, when used as liquid chromatography chiral stationary phases (CSPs), was evaluated using heptane and either 2-propanol or chloroform as organic mobile-phase modifiers. A large variety of racemic compounds have been separated successfully on these CSPs (mainly pharmaceuticals and herbicides). Optimization of these separations was discussed in terms of mobile-phase composition and structural patterns of the injected analytes. The efficiencies of the three prepared materials were compared to those of previously described perphenylated-beta-cyclodextrin column and to analogous cellulose derivative-based CSPs.

Extraction and determination of trinitrotoluenes and products of their biotransformation in soil samples

The separation of a mixture of trinitrotoluenes (TNTs) and their metabolites by reverse phase liquid chromatography using different columns with octadecyl stationary phase is presented. The retention behaviour of a mixture of TNTs and their metabolites was studied under different chromatographic conditions. The mixture of methanol/water was used as a mobile phase in isocratic and gradient modes. The effect of content of organic modifier and temperature of mobile phase has been investigated. Very good RP-HPLC separation of 14 nitroaromatic compounds was obtained using stepped gradient elution. Different properties of extraction procedures for isolation of trinitrotoluenes and their metabolites from soil were tested. Experimental conditions of Soxhlet warm extraction (extractant type, extraction time) suitable for efficient extraction of individual analytes were studied. Recoveries over 80% for all studied compounds and over 90% for almost all (11 compounds) were obtained in short extraction time (1 hour). The formation of precipitate during the Soxhlet extraction was observed and the further treatment of extract was necessary. The amount of precipitate formed from an artificial soil was higher than that from a natural one. THF was found as the best solvent suitable for total dissolution of precipitate.

Doubly tethered tertiary amide linked and ionically bonded diproline chiral stationary phases

Oligoproline chiral stationary phase (CSP) is a new class peptide chiral stationary phase. Many proline chiral stationary phases with different proline chain lengths and linkers have been prepared and evaluated. However, the doubly tethered and ionic type linkers have not been adequately investigated. In this study, covalently and ionically bonded chiral stationary phases with doubly tethered linker were prepared and characterized. The new covalently bonded doubly tethered diproline CSP was applied successfully to resolve various enantiomers of acidic, basic, and neutral compounds with phenyl, naphthyl, anthryl, or similarly sized groups. The enantiorecognition performances of singly and doubly tethered diproline CSPs were comparable. Variation of the type and content of organic modifiers in hexane or heptane mobile phase showed that the branch alcohols such as 2-propanol and 2-butanol, 1,2-dichloroethane, methyl tert-butyl ether, and ethyl acetate in the mobile phase enhanced chiral separation. End-capping on doubly tethered diproline CSP did not always improve the separation factor and resolution. Due to the rigid structure of the double tether, the enantioseparation ability of ionically bonded diproline CSP was well expressed to some analytes.

Study of influence of additives of tyloxapol on the chromatographic characteristics of the model compounds: the comparative characterization of micellar mobile phases of tyloxapol and Triton X‐100

The chromatographic behavior of model compounds of biomedical significance (organic acids, amino acids, drugs) was investigated using mobile phases modified with tyloxapol. The influence of factors such as concentration of tyloxapol, content of organic modifier and pH of mobile phase on the retention factor of solutes was studied. The results were compared with the data obtained by elution with mobile phases containing Triton X-100 additives, since units of Triton X-100 are repeated in the structure of tyloxapol. Divergence in chromatographic behavior of model compounds was explained by the difference in physico-chemical characteristics (microviscosity, polarity, critical micelle concentration, shape of micelles, etc.) of tyloxapol and Triton X-100 micelles.

Displacement effect during HPLC preparative purification of human insulin

HPLC plays a key role in the preparative purification of human insulin. A21-desamidoinsulin is one of the impurities that possesses the chromatographic behavior similar to that of insulin and hence separation from this by-product is rather difficult at the process scale. During the optimization of insulin reversed-phase HPLC purification, when a column was sufficiently overloaded, the effect of displacement of A21-desamidoinsulin molecules from active groups of sorbent by insulin ones was observed. It was suggested that monocarboxylic acid and organic modifier in mobile phase are responsible for the esterification during which the formed ester promotes the displacement effect. This effect was studied in order to optimize the purification of human insulin at the process scale.

Prediction of supercritical fluid chromatographic retention factors at different percents of organic modifiers in mobile phase

In this work quantitative structure-retention relationship models were developed to predict the solute retention factors in supercritical fluid chromatography (SFC) in various organic modifiers. Data set contains the retention data of 35 various organic compounds in 0, 2, 4 and 6% of methanol in mobile phase. The obtained 140 data points were divided into training, internal and external test sets which have 93, 23 and 24 retention data. The diversity validation test was performed on data the set to ensure that the structure of the training and/or test sets can represent those of the whole ones. Descriptors which were selected by stepwise multiple linear regression (MLR) methods are: the percent of organic modifier in mobile phase, salvation connectivity index chi-2, salvation connectivity index chi-5, H attached to heteroatom, the 2(nd) structural information content and polarity parameter. These descriptors were used as features in generation of linear and non-linear models using MLR and artificial neural network (ANN) methods, respectively. The root mean square error of MLR model are 0.116, 0.138 and 0.260 for training, internal and external test sets, respectively, while these values are 0.036, 0.097 and 0.244 for ANN model, respectively. Comparison between these values and other statistical parameters obtained from these two models reveals the credibility of ANN in prediction of solute retention factors in SFC.

Liquid chromatographic resolution of vigabatrin and its analogue γ-amino acids on chiral stationary phases based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid

Racemic vigabatrin, an anticonvulsant drug used for the treatment of epilepsy, and its analogue γ-amino acids were resolved without derivatization on liquid chromatographic chiral stationary phases (CSPs) based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid. Between the two CSPs which contain 3 methylene-unit or 11 methylene-unit spacer group, the latter was found to be greater than the former in the resolution of vigabatrin and its analogue γ-amino acids, the separation, α, and the resolution factor, R S, for the resolution of vigabatrin on the latter being 1.91 and 4.57, respectively. The chromatographic behaviors for the resolution of vigabatrin and its analogue γ-amino acids on the two CSPs were found to be dependent on the type and the content of organic and acidic modifiers in aqueous mobile phase.

Resolution of β‐amino acids on a high performance liquid chromatographic doubly tethered chiral stationary phase containing N–CH3 amide linkage based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid

A doubly tethered chiral stationary phase (CSP) containing N-CH(3 )amide linkage based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was applied to the resolution of various beta-amino acids. The chiral recognition behaviors for the resolution of beta-amino acids on the doubly tethered CSP were consistent with those on the corresponding singly tethered CSP while the chiral recognition ability of the doubly tethered CSP was generally greater in terms of both the separation (alpha) and the resolution factors (R(S)) than that of the corresponding singly tethered CSP. From these results, it was concluded that attaching the second tethering group to silica gel through a carbon atom of the first tethering group of the CSP improves the chiral recognition ability for the resolution of beta-amino acids without any change in the chiral recognition mode. The retention factors (k(1)) on the doubly tethered CSP were larger than those on the corresponding singly tethered CSP and these retention factors were found to be controllable with the variation of the type and the content of the organic and/or acidic modifier in the aqueous mobile phase without significant change in the separation and the resolution factors.

Liquid chromatographic direct resolution of β‐amino acids on a doubly tethered chiral stationary phase containing NH amide linkage based on (+)‐(18‐crown‐6)‐ 2,3,11,12‐tetracarboxylic acid

A doubly tethered chiral stationary phase (CSP) prepared by bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to doubly tethered primary aminoalkyl silica gel was used for the resolution of various beta-amino acids. All the beta-amino acids tested were resolved quite well, the separation (alpha) and the resolution factors (RS) being in the ranges 1.34-2.09 and 2.52-7.45, respectively, with a mobile phase of methanol-water (50:50, v/v) containing 10 mM acetic acid. The chiral recognition efficiency of the doubly-tethered CSP was found to be generally superior to that of the corresponding singly-tethered CSP in the resolution of beta-amino acids. The chiral recognition behaviors for the resolution of beta-amino acids on the doubly tethered CSP were examined by varying the type and content of organic and acidic modifiers in the aqueous mobile phase and the column temperature.

Effects of elevated temperature and mobile phase composition on a novel C18 silica column

A novel polydentate C18 silica column was evaluated at an elevated temperature under acidic, basic, and neutral mobile phase conditions using ACN and methanol as the mobile phase organic modifier. The temperature range was 40-200 degrees C. The mobile phase compositions were from 0 to 80% organic-aqueous v/v and the mobile phase pH levels were between 2 and 12. The maximum operating temperature of the column was affected by the amount and type of organic modifier used in the mobile phase. Under neutral conditions, the column showed good column thermal stability at temperatures ranging between 120 and 200 degrees C in methanol-water and ACN-water solvent systems. At pH 2 and 3, the column performed well up to about 160 degrees C at two fixed ACN-buffer compositions. Under basic conditions at elevated temperatures, the column material deteriorated more quickly, but still remained stable up to 100 degrees C at pH 9 and 60 degrees C at pH 10. The results of this study indicate that this novel C18 silica-based column represents a significant advancement in RPLC column technology with enhanced thermal and pH stability when compared to traditional bonded phase silica columns.

Effect of temperature on gradient reequilibration in reversed-phase liquid chromatography

The effect of mobile phase modifier and temperature on gradient reequilibration is examined using three different stationary phases. The stationary phases studied are a traditional C 18 phase, a polar endcapped C 18 phase, and an alkyl phase with a polar embedded group. It was observed that both temperature and choice of mobile phase organic modifier had an effect on gradient reequilibration volume on both the traditional C 18 stationary phase and the polar endcapped phase. On both these phases, at any given temperature, the reequilibration volume was generally smaller when methanol was used as the mobile phase modifier as compared to acetonitrile. As the temperature is increased from 10 to 50 °C, significant reductions in reequilibration volume were observed with both mobile phase modifiers. In contrast, neither temperature nor choice of modifier appeared to have much effect on reequilibration volume when the polar embedded group stationary phase was considered.

An unexpected observation concerning the effect of anionic additives on the retention behavior of basic drugs and peptides in reversed-phase liquid chromatography

Anionic species with ion pair forming ability are commonly used to enhance the retention and efficiency of basic analytes in RPLC separations. However, little is known about the interactions between organic mobile phase modifiers and such ion pairing anions. In this work, we measured the magnitude of the retention increase of basic drugs and peptides upon addition of strong inorganic ion pairing anions (e.g. perchlorate) as a function of the volume fraction of modifier in acidic water-acetonitrile mobile phases on two different stationary phases. We found that the increase in retention upon addition of various salts depended strongly on the eluent strength. In general, larger retention increases upon addition of the anion were observed at higher organic fractions. Regression of retention against the volume fraction of organic modifier indicated that the ion pair forming anions substantially decreased S values while only slightly changing ln k ′ w values. The decrease in S is the major cause of the retention increase of basic drugs and peptides when such anions are added to the mobile phase.

Liquid Chromatographic Chiral Separations by Crown Ether‐Based Chiral Stationary Phases

Liquid chromatographic chiral stationary phases (CSPs) based on chiral crown ethers have been successfully utilized in the separation of the enantiomers of various racemic compounds containing a primary amino group. Especially, CSPs based on chiral crown ethers incorporating chiral binaphthyl unit or tartaric acid unit have been most successful. In this review, the development of CSPs based on chiral crown ethers incorporating a chiral binaphthyl unit or a tartaric acid unit, their applications to the resolution of various primary and non‐primary amino compounds with the variation of the type and the content of organic, acidic, and inorganic modifiers in an aqueous mobile phase or with a non‐aqueous mobile phase and the efforts to improve the chiral recognition efficiency or the stability of the CSPs have been discussed.

Solvent and temperature gradients in separation of synthetic oxyethylene‐oxypropylene block (co)polymers using high‐temperature liquid chromatography

Chromatographic behavior of synthetic block (co)oligomer samples (EO)n(PO)m(EO)n and (PO)n(EO)m(PO)n with different distribution of propylene oxide (PO) and ethylene oxide (EO) monomer units was investigated on three types of stationary phases on zirconium dioxide support: Zr-PS (polystyrene), Zr-carbon, and Zr-carbon C18. The effects of the distribution and sequence of the oxyethylene and oxypropylene monomer units on the chromatographic retention depend on the type of the stationary phase, but are strongly affected by the organic modifier (methanol or ACN) in aqueous-organic mobile phase. Special attention was focused on the influence of the mobile-phase composition on the separation according to the EO and PO distribution. Zirconia-based columns are stable at elevated temperatures and can be used in high-temperature LC (HTLC); hence, we investigated the temperature effects on the chromatographic behavior up to 90 degrees C. The applications of solvent and temperature gradients were compared on the zirconia stationary phases in the RP mode.

Preparation of a new doubly tethered chiral stationary phase based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid and its application

A new doubly tethered chiral stationary phase (CSP) based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was prepared by attaching the second tethering group to silica gel through a carbon atom of the first tethering group of the corresponding singly tethered CSP, which was previously developed in our laboratory. The new doubly tethered CSP was applied successfully to the resolution of various racemic α-amino acids, amines and amino alcohols containing a primary amino group. In most cases, the chiral recognition efficiency of the new doubly tethered CSP was superior to that of the corresponding singly tethered one in the resolution of α-amino acids, amines and amino alcohols. In the resolution of some racemic primary amino compounds, the new doubly tethered and the corresponding singly tethered CSPs were complementary with each other. The chiral resolution behaviors on the new doubly tethered CSP were examined with the variation of the type and content of organic and acidic modifiers in aqueous mobile phase and the column temperature. The chiral resolution behaviors on the new doubly tethered CSP were generally quite similar to those on the corresponding singly tethered CSP. The stability of the new doubly tethered CSP was greater than that of the corresponding singly tethered CSP.

A Stepwise Stoichiometric Representation To Confirm the Dependence of Pesticide/Humic Acid Interactions on Salt Concentration and To Test the Performance of a Silica Bonded Humic Acid Column

In a previous paper (André et al., in press), a novel chromatographic column was developed in our laboratory for studying the binding of pesticides with humic acid (HA), the main organic component in soil. It was demonstrated that this column supported a low fraction of organic modifier in the aqueous mobile phase (<0.25 (v/v)). To overcome this limitation for a practical use, a column in which the stationary phase was based on silica gel with chemically bonded humic acid was created. It was shown that this novel HA column supported a higher methanol fraction (<0.55 (v/v)). As well, the dependence of pesticide/humic acid interactions on salt (sodium chloride) concentration has been expressed in terms of a stepwise stoichiometric representation, which leads to a specific equation for the partition of the added salt between the pesticide molecule, the HA, and the pesticide/HA complex. Based on this novel equation, the dependence of the pesticide/humic acid association on the salt concentration can be formulated via a relation similar to the one of Tanford. In addition, for the first time, the calculation of the affinity energy distribution for different values of the salt concentration in the mobile phase confirmed the existence of several types of binding sites on the HA macromolecule.

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 × 4.6 mm I.D.) and second dimension (2nd-D) (5 cm × 4.6 mm 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 (CH 3OH) 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 60 s. 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.

Unusual chromatographic behavior of oligonucleotide sequence isomers on two different anion exchange HPLC columns

The retention behavior of the unmodified phosphodiester oligonucleotide sequence isomers was investigated on two different anion exchange columns: Biospher GMB 1000Q (based on DEAE-modified glycidyl methacrylate) and PolyWAX LP (based on silica with a crosslinked coating of linear polyethyleneimine). There was a notable difference in retention of oligonucleotides of the same composition but differing in the position of a single base. The most pronounced difference was observed between the oligonucleotides with the variable base in the end and in the center of the sequence. The use of either acetonitrile or 2-propanol as a mobile phase organic modifier did not markedly affect the retention time patterns. Prediction of the retention times of oligonucleotides must take into account the base position as well as identity. This is the first report of such a “same composition different sequence” effect, described for the short peptides, for synthetic oligonucleotides.

Development of acrylate-based monolithic stationary phases for electrochromatographic separations

Organic monolithic stationary phases were synthesized in fused-silica capillaries. They were prepared by in situ polymerization under UV irradiation of various alkyl acrylates, 1,3-butanediol diacrylate, and 2-acrylamido-2-methyl-1-propanesulfonic acid in a ternary porogenic solvent. The resulting stationary phases were tested in CEC. The influence of UV irradiation energy on the resulting separative performances of the monoliths was studied. It was thus demonstrated that the use of hexyl acrylate rather than butyl acrylate and lauryl methacrylate gives highly efficient monoliths (more than 300 000 plates per meter) with optimized EOF. It was also confirmed that the mobile phase ionic strength may affect significantly the separation efficiency. The influence of the nature of the mobile phase organic modifier (ACN or methanol) on EOF, retention, efficiency, and selectivity was studied and differences were observed. Finally, the performances of monolithic stationary phases developed and optimized for CEC separations were evaluated in nanoLC.

Retention Behavior of Oligonucleotides on a Glycidyl Methacrylate-Based DEAE-Modified Sorbent

The retention behavior of unmodified phosphodiester mixed-sequence oligonucleotides has been investigated under different conditions on a glycidyl methacrylate-based, DEAE-modified anion-exchange Biospher GMB 1000Q column. It was verified that separation is based not only on an anion-exchange mechanism but also on secondary interactions of the bases with the stationary phase. Notably different retention was observed for oligonucleotides differing only in the position of one base in the sequence. The nature of these secondary interactions and the effect of sequence on retention is discussed. The effect on retention of the type and concentration of organic mobile phase modifier, mobile phase pH, sample molecular weight, and the bases in the sample was investigated. A linear relationship was found between retention and the molecular weight of mixed-sequence oligonucleotides with lengths from 15 to 40. Resolution of N − 1 synthetic by-products from the full-length product was also evaluated.