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1.
J Chromatogr A ; 1692: 463820, 2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-36796276

RESUMO

Typical chromatographic analysis of chiral compounds requires the use of achiral methods to evaluate impurities or related substances along with separate methods to evaluate chiral purity. The use of two-dimensional liquid chromatography (2D-LC) to support simultaneous achiral-chiral analysis has become increasingly advantageous in the field of high-throughput experimentation where low reaction yields or side reactions can lead to challenging direct chiral analysis. Advancements in multi-dimensional chromatography have led to the development of robust 2D-LC instrumentation with reversed phase solvent systems (RPLC-RPLC) enabling this simultaneous analysis, eliminating the need to purify crude reaction mixtures to determine stereoselectivity. However, when chiral RPLC cannot separate a chiral impurity from the desired product, there are few viable commercial options. The coupling of NPLC to RPLC (RPLC-NPLC) continues to remain elusive due to solvent immiscibility between the two solvent systems. This solvent incompatibility leads to lack of retention, band broadening, poor resolution, poor peak shapes, and baseline issues in the second dimension. A study was conducted to understand the effect of various water-containing injections on NPLC and applied to the development of robust RPLC-NPLC methods. Following thoughtful consideration and modifications to the design of a 2D-LC system in regards to mobile phase selection, sample loop sizing, targeted mixing, and solvent compatibility, proof of concept has been demonstrated with the development of reproducible RPLC-NPLC 2D-LC methods to perform simultaneous achiral-chiral analysis. Second dimension NPLC method performance proved comparable to corresponding 1D-NPLC methods with excellent percent difference in enantiomeric excess results ≤ 1.09% and adequate limits of quantitation down to 0.0025 mg/mL for injection volumes of 2 µL, or 5 ng on-column.


Assuntos
Cromatografia de Fase Reversa , Projetos de Pesquisa , Cromatografia de Fase Reversa/métodos , Solventes/química , Água , Estereoisomerismo
2.
Anal Chem ; 94(46): 16142-16150, 2022 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-36356979

RESUMO

Two-dimensional liquid chromatography (2D-LC) is a powerful technique used to characterize complex samples such as synthetic polymers, biomacromolecules, and hybrid modalities (conjugates, oligonucleotides, nanoparticles, etc., which fall between traditional small molecules and large molecules). Characterizing such molecules often requires a highly orthogonal 2D-LC workflow to resolve structurally similar impurities. However, it remains a challenge to achieve truly orthogonal 2D-LC coupling due to incompatibility of the chromatographic conditions used in each dimension. In this work, we present a facile strategy of connecting an in-line mixer, in-line mixing modulation (ILMM), to realize challenging 2D-LC workflows: (1) coupling gel permeation chromatography (GPC) with reversed-phase liquid chromatography (RPLC) for hydrophobic oligomer analysis and (2) coupling ion-pair reversed-phase (IPRP) with hydrophilic interaction liquid chromatography (HILIC) for polar antisense oligonucleotide (ASO) analysis. Compared with the state-of-the-art commercially available active solvent modulation (ASM), engaging the ILMM significantly reduces the peak distortion in GPC-RPLC, allowing an at least 67% higher transfer volume from the primary to secondary dimension, and resolves the ASO sample breakthrough in selective comprehensive IPRP×HILIC. Also remarkably, ILMM demonstrated superiority in comprehensive RPLC×RPLC analysis in comparison with ASM, suggesting its potential in broader 2D-LC applications. In addition to chromatography improvement, ILMM offers several advantages over benchmark modulation approaches in regard to alleviating the need of an additional dilution flow and a simple as well as flexible system configuration, opening many opportunities to establish innovative and versatile multidimensional workflows for characterizing compounds with increasing complexity.


Assuntos
Cromatografia de Fase Reversa , Oligonucleotídeos , Solventes , Cromatografia de Fase Reversa/métodos , Cromatografia Líquida/métodos , Interações Hidrofóbicas e Hidrofílicas
3.
J Sep Sci ; 35(14): 1748-54, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22807357

RESUMO

In general, chromatographic analysis of chiral compounds involves a minimum of two methods; a primary achiral method for assay and impurity analysis and a secondary chiral method for assessing chiral purity. Achiral method resolves main enantiomeric pairs of component from potential impurities and degradation products and chiral method resolves enantiomeric pairs of the main component and diastereomer pairs. Reversed-phase chromatographic methods are preferred for assay and impurity analysis (high efficiency and selectivity) whereas chiral separation is performed by reverse phase, normal phase, or polar organic mode. In this work, we have demonstrated the use of heart-cutting (LC-LC) and comprehensive two-dimensional liquid chromatography (LC × LC) in simultaneous, sequential achiral and chiral analysis and quantitation of minor, undesired enantiomer in the presence of major, desired enantiomer using phenylalanine as an example. The results were comparable between LC-LC and LC × LC with former offering better sensitivity and accuracy. The quantitation range was over three orders of magnitude with undesired D-phenylalanine detected at approximately 0.3% in the presence of predominant, desired L-phenylalanine (99.7%). The limit of quantitation was comparable to conventional high-performance liquid chromatography. A reversed-phase C18 achiral column in the primary and reversed-phase Chirobiotic Tag chiral column in the secondary dimension were used with a compatible mobile phase.


Assuntos
Cromatografia Líquida/métodos , Fenilalanina/química , Cromatografia Líquida/instrumentação , Estereoisomerismo
4.
J Sep Sci ; 29(4): 510-8, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16583689

RESUMO

A comprehensive 2-D-LC-MS method has been developed by coupling columns of different selectivity. The primary column eluate is alternately trapped and sampled onto the secondary columns through a guard column interface. When one guard column traps the eluate, the other injects the previously trapped components onto a secondary column. This cycle is repeated throughout the chromatogram. The use of dual secondary columns provides the secondary columns with additional time to generate high-speed chromatograms. Each secondary column generates alternate chromatograms which when combined generate the entire chromatogram. The primary column separation is comparable to conventional HPLC, whereas the secondary column separation is fast. With both the columns operating in reverse phase mode, one would expect strong correlation in the two-dimensional retention and hence inefficiency in separation. However, differences in column operation modes, interaction mechanisms, and vendor silica result in a complementary separation. The system was evaluated by comparing it to one-dimensional counterparts and coupled column chromatography. Although some correlations were observed in 2-D-LC-MS, peaks do show two-dimensional distribution with superior UV and MS data as co-elution is minimized. Also, the ease of converting conventional systems to 2-D-LC-MS is discussed.


Assuntos
Cromatografia Líquida , Espectrometria de Massas , Cromatografia Líquida/instrumentação , Cromatografia Líquida/métodos , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Teste de Materiais , Estrutura Molecular , Preparações Farmacêuticas/análise
5.
J Chromatogr A ; 1066(1-2): 47-53, 2005 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-15794554

RESUMO

Mixed mode stationary phases with ion-pairing reagent (acidic or basic) as integral part of hydrophobic chain offers unique selectivity, and hence, are ideal for multidimensional separations. The retention of hydrophobic components is a function of organic content, whereas that of charged species is a function of organic content, ionogenic modifier and its level in the mobile phase. Hence, by controlling the parameters influencing component retention (stationary phase and mobile phase), the selectivity of chemical components in the two-dimensional plane can be manipulated to improve the separation. A two-dimensional liquid chromatograph has been developed by coupling similar and dissimilar mixed mode stationary phases in the two dimensions. This technique has immense potential in resolving co-eluting components as the retention mechanism in the two-dimensions are complementary. However, with only part of the primary column eluent sampled into the secondary column, the technique is limited to qualitative analysis.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Fenômenos Químicos , Físico-Química , Cromatografia Líquida de Alta Pressão/instrumentação , Indicadores e Reagentes , Reprodutibilidade dos Testes
6.
Anal Chem ; 75(14): 3484-94, 2003 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-14570201

RESUMO

A simple approach to two-dimensional liquid chromatography has been developed by coupling columns of different selectivity using a 12-port, dual-position valve and a standard HPLC system. The valve at the junction of the two columns enables continuous, periodic sampling (injection) of the primary column eluent onto the secondary column. The separation in the primary dimension is comparable to conventional HPLC, whereas the secondary column separation is fast, lasting several seconds. The high-speed separation in the secondary dimension enables the primary column eluent to be sampled with fidelity onto the secondary column throughout the chromatographic run. One might expect a coupled column liquid chromatography system operating in reverse-phase mode to be strongly correlated and, hence, inefficient. However, by applying a solvent gradient in the primary dimension and by progressively incrementing the solvent strength in the secondary dimension (tuning), the inefficiency or cross correlation between the two dimensions is minimized. In a tuned two-dimensional system, the influence of primary column retention (usually hydrophobicity) is minimal on secondary column retention. This enables subtle differences in component interaction with the two stationary phases to dominate the secondary column retention. The peaks are randomly dispersed over a retention plane rather than along a diagonal, resulting in an orthogonal separation. The peak capacity is multiplicative, and each component has a unique pair of retention times, enabling positive identification. In addition, the location of the component provides two independent measures of molecular properties. The 2D-LC system was evaluated by analyzing a test mixture made of some aromatic amines and non-amines on different secondary columns (ODS-AQ/ODS monolith, ODS/amino, ODS/cyano). The relative location of sample components in the two-dimensional plane varied significantly with change in secondary column. Among the secondary columns, the amino and cyano columns offered the most complementary separation, with the retention order of several components reversed in the secondary dimension. The theoretical peak capacity of the 2D-LC system was around 450 for a separation lasting 30 min. A 2D-LC system involving amino and cyano columns resulted in a high-speed separation of the test mixture, with most of the chemical components resolved within a few minutes.

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