Separation Of Positional Isomers Research Articles

Micellar enhanced chromatographic separation of selected hazardous chemical present in hair dye and their detection in formulations and swab, including assessment of damage caused to cuticle of hair shaft

Hydroquinone (HQ), resorcinol (RS), m-aminophenol (m-AMP) and p-phenylenediamine (p-PPD) are aromatic compounds which are generally used in hair dyes to provide different colours to hair. In European Union the concentrations of HQ, RS, m-AMP and p-PPD is regulated in hair dyes and other cosmetic products by EU commission regulation EU/2019/831. This legislation is generally exercised because all these compounds are toxic and may cause severe allergies when used regularly. However in India no such regulations exist to monitor these toxic compounds in hair dyes therefore in this study a simple, rapid, economical and ecofriendly micellar liquid chromatographic (MLC) technique has been developed which can monitor all the selected toxic compounds simultaneously. HQ and RS are positional isomers and are difficult to be separated by HPLC whereas with the developed MLC method it was well separated and detected. The developed MLC technique has been applied to detect and quantify selected analytes in oxidative and non-oxidative hair dyes and swab samples from the scalp. The simultaneous separation of selected analytes was performed in mobile phase 0.09 M SDS, 0.01 M NaH2PO4–2% v/v 1-butanol at pH 7 running through C18 column under isocratic mode at 1 mL/min. flow rate. All the analytes were eluted within 6 min. The present method has been validated following the EURCHEM Guideline, 2014 in terms of calibration range (0.08–15 µg/mL), limit of detection (0.01–0.09 µg/mL), limit of quantification (0.08–0.35 µg/mL), accuracy (<5.6%), precision (91–105%) and robustness (<5.8%). The selected compounds in hair dye formulation were found in the range of 0.06–12.2 µg/mL (when diluted 25 times). Hair dyes persistence study was conducted up to 10 days from the day of application on the scalp, suggesting that the dyes were not completely washed off and were retained on the scalp for more than one week. SEM analysis of dyed hair revealed that hair are severely damaged due to use of dyes. The advantage of the developed method is that it could easily be adopted by quality control and cosmetic laboratories for quality control and check for the simultaneous separation of positional isomers together with two other aromatic compounds.

Single-Crystalline Covalent Organic Frameworks as High-Performance Liquid Chromatographic Stationary Phases for Positional Isomer Separation

Vigorously developing new stationary phases to meet the requirements for the separation of positional isomers that have similar physicochemical properties is still an urgent topic in separation science. Herein, a single-crystalline covalent organic framework (COF-300) packed column for the separation of positional isomers in high-performance liquid chromatography (HPLC) was reported for the first time. Benefitting from its regular shape, excellent chemical and thermal stability, microporous feature, and strong hydrophobicity of single-crystalline COF-300, the single-crystalline COF-300-packed column showed excellent resolution for the separation of positional isomers, including nitroaniline, dichlorobenzene, dibromobenzene, diiodobenzene, diethylbenzene, chloronitrobenzene, bromonitrobenzene, and iodonitrobenzene isomers, which cannot be all separated on commercial columns and a polycrystalline COF-300-packed column. Especially, the resolution values for m-/p-diiodobenzene and o-/m-diiodobenzene were 4.45 and 2.53. Moreover, the alkylbenzene, monosubstituted aromatics, polycyclic aromatic hydrocarbons, and the mixture of ethylbenzene and styrene were also baseline separated on the single-crystalline COF-300-packed column. This successful application not only confirmed the great potential of single-crystalline COFs in HPLC separation of positional isomers but also pioneered the utilization of single-crystalline COFs in separation science.

Chiral polyaniline modified Metal-Organic framework Core-Shell composite MIL-101@c-PANI for HPLC enantioseparation

Metal-organic frameworks (MOFs) have attracted a substantial amount of attention due to special properties, such as gas adsorption, chromatographic separation, and catalytic activity. Herein, a post-modification method was used to modify the surface of achiral MOFs using chiral polyaniline to synthesize a chiral core–shell composite (MIL-101@c-PANI), which was used for the first time as a stationary phase for the separation of chiral compounds. Twelve kinds of chiral compounds, including alcohols, ketones, esters, aldehydes, organic acids and amines were separated on the MIL-101@c-PANI packed column. Moreover, the column also showed good selectivity for the separation of positional isomers. Some chiral compounds that cannot be separated using a commercially available tris(3,5-dimethylphenylcarbamoyl) amylose-packed column (AD column), could be separated on the MIL-101@c-PANI column. The effects of the analyte mass and column temperature on the separation efficiency were investigated. The column exhibited good stability and reproducibility during the enantioseparation of chiral compounds. The MIL-101@c-PANI composite gives rise to a synergistic effect by combining the advantages of c-PANI and MIL-101, which is favorable for an improvement in the chiral separation performance. Therefore, this work shows that such composite materials will be very attractive in the field of separation science.

Separation of multiphosphorylated cyclopeptides and their positional isomers by hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI-MS)

Peptides are efficient models used in different fields such as toxicology to study the interactions of several contaminants at the molecular scale, requiring the development of bio-analytical strategies. In this context, Hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI-MS) was used to separate synthetic multiphosphorylated cyclopeptides and their positional isomers at physiological pH. We assessed (i) the selectivity of eleven HILIC columns, from different manufacturers and packed with diverse polar sorbents, and (ii) the effect of mobile phase composition on the separation selectivity. The best selectivity and baseline resolution were achieved with the columns grafted by neutral sorbents amide and diol. Furthermore, we investigated the HILIC retention mechanism of these peptides by examining the effect of the number of phosphorylated residues in the peptide scaffold on their retention. The peptide behavior followed the classical hydrophilic partitioning mechanism exclusively on amide and diol columns. This trend was not fully respected on bare and hybrid silica due to the attractive/repulsive interactions of the deprotonated surface silanol groups with the Arginine or Glutamate residues in the peptide scaffold according to the peptide sequence. The position of the phosphorylated amino acid in the peptide backbone also showed to have an impact on the retention, making possible the separation of positional isomers of these multiphosphorylated cyclic peptides using HILIC.

QSRR modelling aimed on the HPLC retention prediction of dimethylamino- and pyrrolidino-substitued esters of alkoxyphenylcarbamic acid

Two homologous series of alkoxyphenylcarbamic acids esters with biological activities were subjected to the QSRR (Quantitative Structure–Retention Relationships) study. Retention factors of studied derivatives were measured in four different HPLC (High Performance Liquid Chromatography) systems. Experimental data in combination with in silico calculated molecular descriptors were used for development complex QSRR model for prediction of retention factor and partially for elucidation of separation mechanisms. The best results in QSRR modelling were given by artificial neural networks in the terms of developing one single robust model with high accuracy of logk prediction ( $${Q}_{\mathrm{F}3}^{2}$$ = 0.998) for all studied chromatographic systems. Chemometrical techniques were also used to elucidate the separation mechanisms occurring in the HPLC systems. We found similarities between particular systems and we identified systems which were the most suitable for separation of positional isomers of studied compounds. We observed that solvent significantly influenced the chromatographic separation of positional isomers depending on the used column, whereas combination of acetonitrile and C18-phenyl column caused suppression of π − π interactions, so retention was wholly determined by the hydrophobic interactions.

Covalent coupling fabrication of microporous organic network bonded capillary columns for gas chromatographic separation

Microporous organic networks (MONs) have shown great promise in separation science recently. Exploring novel, simple and convenient strategy to fabricate MONs coated capillary columns for gas chromatography (GC) still remains challenging but desirable for the development of MONs in chromatographic separation. To extend the potential application of MONs in separation science and to further develop novel method for the fabrication of MONs-based capillary columns, here we demonstrate a novel covalent coupling strategy to fabricate uniform MONs bonded capillary columns for GC separation of position isomers and hydrocarbons. The bare capillary column was firstly modified with (3-bromopropyl)trimethoxysilane to provide bromine sites for coupling with alkynyl monomers. The amino- and hydroxyl-functionalized MONs (MON-NH2 and MON-OH) were then directly grown onto the inner wall of the brominated capillary columns via the covalent coupling between bromine and alkynyl groups. The uniform MON-NH2 and MON-OH bonded capillary columns were obtained and showed good resolution for GC separation of dichlorobenzene, chlorotoluene, bromotoluene, and propylbenzene position isomers and many other hydrocarbons including linear alkanes, alkylbenzenes, pinene isomers, cyclohexane and benzene, ketones and aldehydes. The MONs bonded capillary columns also owned good lifetime and precision for dichlorobenzene isomers with the relative standard deviations (RSDs) of 0.2–0.3%, 1.2–2.1%, and 1.7–2.5% for retention time, peak height and peak area, respectively. In addition, the fabricated MON-NH2 and MON-OH bonded capillary columns offered better resolution than commercial InertCap-1, InertCap-5, InertCap-1701 and InertCap-WAX capillary columns for the separation of chlorotoluene and bromotoluene position isomers. These results revealed the feasibility of covalent coupling strategy to fabricate MONs-based stationary phases in GC, highlighting the potential of MONs in separation science.

New approach to prepare fluorogenic branched dextrins for assaying glycogen debranching enzyme.

Glycogen debranching enzyme (GDE), together with glycogen phosphorylase (GP), is responsible for the complete degradation of glycogen. GDE has distinct catalytic sites for 4-α-glucanotransferase and amylo-α-1,6-glucosidase. For the GDE sensitive assay, we previously developed the GP limit fluorogenic branched dextrin Glcα1-4Glcα1-4Glcα1-4Glcα1-4(Glcα1-4Glcα1-4Glcα1-4Glcα1-6)Glcα1-4Glcα1-4Glcα1-4GlcPA (B4/84, where Glc = D-glucose and GlcPA = 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol). However, B4/84 is not widely available because of difficulties in its chemical synthesis and positional-isomer separation (0.33% yield by α-1,6-coupling of maltotetraose with Glc7-GlcPA). In this study, we attempted to develop an efficient method for the preparation of Glcα1-4Glcα1-4Glcα1-4Glcα1-4(Glcα1-4Glcα1-4Glcα1-4Glcα1-6)Glcα1-4Glcα1-4GlcPA (B3/74), which was designed to have the minimum essential dextrin structure for GDE. First, Glcα1-6Glcα1-4Glcα1-4GlcPA (B3/31) was prepared from commercially available Glcα1-6Glcα1-4Glcα1-4Glc. Using α-cyclodextrin as a donor substrate, cyclodextrin glucanotransferase elongated both the main and side branches on B3/31, while all the glycosidic bonds in B3/31 were left intact. After exhaustive digestion with GP, B3/74 was obtained from B3/31 with 16% yield, a value that is 48-fold greater than that previously reported for B4/84. GDE 4-α-glucanotransferase exhibited high activity toward both B3/74 and B4/84. In addition, we studied the efficient conversion of B3/74 into Glcα1-4Glcα1-4Glcα1-4Glcα1-4(Glcα1-6)Glcα1-4Glcα1-4GlcPA (B3/71), which has the best dextrin structure for the GDE amylo-α-1,6-glucosidase.

Capillary Zone Electrophoresis-Top-Down Tandem Mass Spectrometry for In-Depth Characterization of Hemoglobin Proteoforms in Clinical and Veterinary Samples.

Hemoglobin (Hb) constitutes an important protein in clinical diagnostics-both in humans and animals. Among the high number of sequence variants, some can cause severe diseases. Moreover, chemical modifications such as glycation and carbamylation serve as important biomarkers for conditions such as diabetes and kidney diseases. In clinical routine analysis of glycated Hb, sequence variants or other Hb proteoforms can cause interference, resulting in wrong quantification results. We present a versatile and flexible capillary zone electrophoresis-mass spectrometry screening method for Hb proteoforms including sequence variants and modified species extracted from dried blood spot (DBS) samples with virtually no sample preparation. High separation power was achieved by application of a 5-layers successive multiple ionic polymer layers-coated capillary, enabling separation of positional isomers of glycated α- and β-chains on the intact level. Quantification of glycated Hb was in good correlation with the results obtained in a clinical routine method. Identification and characterization of known and unknown proteoforms was performed by fragmentation of intact precursor ions. N-Terminal and lysine glycation could be identified on the α- and β-chain, respectively. The versatility of the method was demonstrated by application to dog and cat DBS samples. We discovered a putative new sequence variant of the β-chain in dog (T38 → A). The presented method enables separation, characterization, and quantification of intact proteoforms, including positional isomers of glycated species in a single run. Combined with the simple sample preparation, our method represents a valuable tool to be used for deeper characterization of clinical and veterinary samples.

A hydroxyl-functionalized homochiral porous organic cage for gas chromatographic separations

A hydroxyl-functionalized homochiral porous organic cage (POC) was synthesized and characterized by FTIR, NMR, thermogravimetric analysis (TGA), MALDI-TOF-MS, and elemental analysis. The synthesized homochiral POC was used as stationary phase to prepare a capillary gas chromatography (GC) column by a static coating method. The fabricated column shows excellent selectivity not only for the separation of positional isomers but also for the resolution of various racemates. Thirty-nine racemates have been resolved on the column, including alcohols, diols, halohydrocarbons, epoxides, esters, lactones, ketones, ethers, and organic acids. Compared to the commercial β-DEX 120 column and previously reported chiral POCs (CC3-R, CC9, and CC10)-coated columns, there are 11, 10, 24, and 15 tested racemates thatcannot be resolved on β-DEX 120 column, CC3-R column, CC9 column, and CC10 column, respectively. This reveals that the fabricated column has prominent complementarity or superior separation performance to these columns in enantioseparation. Besides, the fabricated column can achieve some enantioseparations which are notpossible using all previously reported chiral POC-based columns. Some positional isomers (xylenes, dichlorobenzenes, dibromobenzenes, nitrochlorobenzenes, and nitrobromobenzenes) were alsoseparated with high-resolution values. The column exhibits good repeatability, reproducibility, and stability. The relative standard deviation (RSD) values of retention times were 0.03-0.18%, 0.11-0.92%, and 2.1-6.6% for run-to-run (n = 5), day-to-day (n = 5), and column-to-column (n = 3), respectively. The experimental results demonstrate the great potential of POCs for practical application in GC. Graphical Abstract A hydroxyl-functionalized homochiral porous organic cage was used as stationary phase for gas chromatography separation of racemates and positional isomers. The resolution of racemates mainly depended on hydrogen bonding, π-interaction, host-guest inclusion, steric fit, etc., while separation of positional isomers by shape-selective guest binding.

Triptycene-based stationary phases for gas chromatographic separations of positional isomers

This work presents the investigation of two triptycene-based materials (TP-3OB and TP-3Im) as the stationary phases for gas chromatographic (GC) separations. The TP-3OB and TP-3Im capillary columns fabricated by static coating exhibited column efficiency of 3000–3500 plates/m for n-dodecane at 120 °C. Also, their McReynolds constants and Abraham system constants were determined to characterize their polarity and molecular interactions with analytes. On the basis of the unique 3D TP architecture, the TP-3OB and TP-3Im stationary phases exhibited complementary high-resolution performance for analytes of a wide ranging polarity, including alkylbenzenes, alkylnaphthalenes, halobenzenes, phenols and anilines, respectively. Moreover, the TP-based columns exhibited good repeatability and reproducibility on the retention times of analytes with the relative standard deviation (RSD) values in the range of 0.01–0.14% for run-to-run, 0.11–0.47% for day-to-day and 0.68–4.7% for column-to-column, respectively. Additionally, their applications for the determination of isomer impurities in the commercial reagents of o-dichlorobenzene, p-/m-diethylbenzene, o-toluidine and 2,3-/3,5-xylidine proved their good potential for practical analysis. This work demonstrates the promising future of the triptycene-based stationary phases for chromatographic separations.

Small alkyl amines as ion-pair reagents for the separation of positional isomers of impurities in phosphate diester oligonucleotides

In an effort to improve separation of impurities in oligonucleotide drugs, alkyl amines of different length and carbon content were evaluated as reagents in ion pair-reversed phase (IP-RP) HPLC with mass spectrometric detection. A range of columns was tested in combination with different buffers, ion-pair modifiers and varying pH adjustments. For phosphorothioate oligonucleotides, larger amines, like tributyl and hexyl amine provided the best chromatography, as small amines tended to broaden peaks due to the separation of diastereoisomers. For phosphate diester oligonucleotides, the best separations were obtained using small alkyl amines, like propyl-, isopropyl- and diethylamine. Conditions optimized for oligonucleotide sequence and type of impurity enabled full separation of the individual components of composite impurities, such as n-1, N3-(2-cyanoethyl)thymine (CNET), deaminated and 3-(2-oxopropyl)imidazopyrimidinone (OPC) impurities. The addition of long-chain alkyl acids like hexanoic acid to the IP buffer resulted in further improvements in peak separation.

Glycol Diglycidyl Ether and γ-Aminobutyric Acid Functionalized Silica as a Mixed Mode Stationary Phase for Capillary Liquid Chromatography

In this study, a mixed mode stationary phase for capillary liquid chromatography (cLC) was prepared by chemical modification of glycol diglycidyl ether (EGDE) and γ-aminobutyric acid (GABA) bonded to the silica, named as Sil–EGDE–GABA. The Sil–EGDE–GABA was characterized by Fourier transform infrared spectroscopy and elemental analysis. The retention of nucleotide bases at different acetonitrile levels in the mobile phase indicated that the stationary phase possessed both hydrophilic chromatography (HILIC) and reversed-phase chromatography (RPLC) characteristics. Positional isomers, aniline compounds and a mixture of polar–nonpolar analytes were separated in RPLC mode. The stationary phase exhibited different separation mechanisms in the separation of positional isomers, compared with a commercial ODS column. Sulfonamides and biogenic amines were successfully separated in HILIC mode. These results demonstrated the application possibilities of the prepared Sil–EGDE–GABA as packing materials in cLC.

Chemoenzymatic Synthesis of N-glycan Positional Isomers and Evidence for Branch Selective Binding by Monoclonal Antibodies and Human C-type Lectin Receptors.

Here, we describe a strategy for the rapid preparation of pure positional isomers of complex N-glycans to complement an existing array comprising a larger number of N-glycans and smaller glycan structures. The expanded array was then employed to study context-dependent binding of structural glycan fragments by monoclonal antibodies and C-type lectins. A partial enzymatic elongation of semiprotected core structures was combined with the protecting-group-aided separation of positional isomers by preparative HPLC. This methodology, which avoids the laborious chemical differentiation of antennae, was employed for the preparation of eight biantennary N-glycans with Galβ1,4GlcNAc (LN), GalNAcβ1,4GlcNAc (LDN), and GalNAcβ1,4[Fucα1,3]GlcNAc (LDNF) motifs presented on either one or both antennae. Screening of the binding specificities of three anti-LeX monoclonal IgM antibodies raised against S. mansoni glycans and three C-type lectin receptors of the innate immune system, namely DC-SIGN, DC-SIGNR, and LSECtin, revealed a surprising context-dependent fine specificity for the recognition of the glycan motifs. Moreover, we observed a striking selection of one individual positional isomer over the other by the C-type lectins tested, underscoring the biological relevance of the structural context of glycan elements in molecular recognition.

Mixed-mode liquid chromatography with a stationary phase co-functionalized with ionic liquid embedded C18 and an aryl sulfonate group

A novel silica stationary phase was designed and prepared through thiol-ene click chemistry by functionalizing imidazolium based ionic liquid embedded C18 and an aryl sulfonate group for the mixed-mode liquid chromatography. The developed stationary phase was characterized by elemental analysis (EA), Fourier transform infrared spectrometry (FT-IR) and thermogravimetric analysis (TGA). The chromatographic performances of the prepared column were investigated by reversed phase chromatography using alkylbenzenes and positional isomers, hydrophilic interaction chromatography using nucleosides, nucleic bases and flavonoids, and ion exchange chromatography using acidic and basic analytes. The stationary phase showed faster separation and better performance for these compounds compared to the conventional alkyl stationary phase, especially for the separation of positional isomers. The investigations about water percentage and pH of the mobile phase for retentions provided information that the stationary phase offered multiple interactions with samples including hydrophobic, hydrophilic and electrostatic interactions. Moreover, improved separation efficiency of Qinghuozhimai tablet and vitamin B2 tablet were successfully achieved respectively on a single mixed-mode column. In conclusion, the multimodal retention capabilities of the developed stationary phase could offer flexible selectivity toward various types of compounds and complex samples.

Separation of positional isomers of nine 2-phenethylamine-derived designer drugs by liquid chromatography-tandem mass spectrometry.

The synthesis of positional isomers of designer drugs is a common way of bypassing legal restrictions. For forensic case work, and especially for the legal assessment of cases, there is a need for screening methods capable of the unequivocal identification of positional isomers. The presented liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method facilitates separation of positional isomers of 9 2-phenethylamine-derived designer drugs in different matrices including seized materials, hair, serum, and urine specimens. Chromatographic separation was achieved on a biphenyl phase using gradient elution with a total runtime of 26minutes. The limit of detection was 25pg/mg for hair samples and ranged from 0.1ng/mL to 0.5ng/mL for serum and from 0.2ng/mL to 1.2ng/mL for urine samples. The method proved to be selective and sensitive and showed good chromatographic resolution (R ≥ 1.2). The method was successfully applied to routine case samples.

A homochiral porous organic cage with large cavity and pore windows for the efficient gas chromatography separation of enantiomers and positional isomers.

Porous organic cages composed of discrete cage molecules have attracted considerable recent attention as gas adsorption materials and separation media. In this study, we report a homochiral porous organic cage CC5 with a large cavity and pore windows as a novel stationary phase for high-resolution gas chromatographic separations. The capillary column was prepared by a static coating method. A large number of racemic compounds have been resolved on the coated capillary column, including derivatized amino acids, alcohols, alcohol amines, esters, ethers, ketones, and epoxides. It is interesting that the CC5-coated capillary column exhibits significant chiral recognition complementarity to a commercial β-DEX 120 column and a previously reported homochiral porous organic cage CC3-R-coated column, which could expand the range of the analytes amenable to separation on porous organic cage-based capillary columns. Moreover, the fabricated column also shows excellent selectivity for the separation of positional isomers, including the challenging ethylbenzene and xylene isomers. Experimental results demonstrate an excellent separation performance and stability of the CC5-coated column, making it promising for gas chromatography applications.

Layer-by-layer preparation of 3D covalent organic framework/silica composites for chromatographic separation of position isomers.

A layer-by-layer approach was developed to prepare 3D covalent organic framework COF-300/silica composites (COF-300@SiO2) with uniform morphology as the stationary phase for high performance liquid chromatography separation of position isomers with high efficiency, selectivity and precision.

Homochiral porous organic cage used as stationary phase for open tubular capillary electrochromatography

Porous organic molecular cages, as a new type of porous materials, have in recent years attracted a tremendous amount of attention for their potential applications. However, to the best of our knowledge, there has been no attempt to utilize porous organic molecular cages as stationary phases in capillary electrochromatography (CEC). We report herein the use of a homochiral porous organic cage (POC) (CC3-R) as a stationary phase in open tubular capillary electrochromatography (OT-CEC) for the separation of chiral compounds and positional isomers. The column was fabricated using CC3-R as the stationary phase by a static coating method. Separation of furoin, benzoin, and alprenlol were achieved on the CC3-R coated column, with the highest resolution value (Rs = 3.35) for the separation of benzoin. The influences of pH and buffer concentration on separation have been investigated. Besides, the CC3-R column also exhibited good selectivity for the separation of positional isomers, including those of nitrophenols, phenylenediamines, aminophenols, and ionones. The run-to-run (n = 5), day-to-day (n = 5), column-to-column (n = 3), and batch-to-batch (n = 3) relative standard deviations (RSDs) for the analyte migration time were in the range of 0.5–1.5%, 0.2–1.8%, 1.2–2.1% and 1.5–2.8%, respectively. The RSDs for the migration time and enantioselectivity of the analyte were less than 5.9% and 2.2% (inter-day, n = 5) after a week of continuous use. This work also indicates that porous organic molecular materials are promising for enantioseparation in CEC and look set to become more attractive in separation science.

Construction of a hydrazone-linked chiral covalent organic framework–silica composite as the stationary phase for high performance liquid chromatography

Covalent organic frameworks (COFs), as an emerging class of crystalline porous organic polymers, have great potential for applications in chromatographic separation owning to their fascinating crystalline structures and outstanding properties. However, development of COF materials as novel stationary phases in high performance liquid chromatography (HPLC) is just in its infancy. Herein, we report the design and construction of a new hydrazone-linked chiral COF, termed BtaMth COF, from a chiral hydrazide building block (Mth) and present a one-pot synthetic method for the fabrication of BtaMth@SiO2 composite for HPLC separation of isomers. The as-synthesized BtaMth chiral COF displays good crystallinity, high porosity, as well as excellent chemical stability. Meanwhile, the fabricated HPLC column by using BtaMth@SiO2 composite as the new stationary phase exhibits high resolution performances for the separation of positional isomers including nitrotoluene and nitrochlorobenzene, as well as cis-trans isomers including beta-cypermethrin and metconazole. Additionally, some effects such as the composition of the mobile phase and column temperature for HPLC separations on the BtaMth@SiO2 packed column also have been studied in detail. The successful applications indicate the great potentials of hydrazone-linked chiral COF–silica composite as novel stationary phase for the efficient HPLC separation.

Theoretical Aspects of Triacylglycerin Positional Isomer Separation on Reverse Phase High Performance Liquid Chromatography.

The purpose of the present study is to bear our hypothesis and to make a profound discussion on the theoretical aspects of triacylglycerin (TG) positional isomer separation on reverse phase high performance liquid chromatography (HPLC). Authentic triacid TG positional isomers and authentic diacid TG positional isomers were injected to a monomeric and polymeric HPLC. Relationship between TG molecular structure and those elution profiles were carefully analyzed. In polymeric column, thickness of the ODS chain is high compared to monomeric column. And for this reason, clearances between the ODS stationary phase chains are considered to be very limited compared to those of monomeric column, thus limiting the TG molecule transformations and rotations to only a "longest stem direction" which gives the smallest resistance in passing through the ODS chains. The longest stem direction of the TG molecule was considered to be the crucial factor for the interaction between the stationary phase. And under this condition, interference occurring between the fatty acid moieties must be the main contributing factor for positional isomer separation in polymeric column. These aspects were considered to explain why they are not consistent with the addition theorem of A.J.P. Martin in partition chromatography.