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Analytical Methods Development, Validation, and Transfer
Published in Sam A. Hout, Manufacturing of Quality Oral Drug Products, 2022
The term reversed phase describes the chromatography mode that is just the opposite of normal phase, namely the use of a polar mobile phase and a non-polar (hydrophobic) stationary phase. Reversed-phase chromatography employs a polar (aqueous) mobile phase (hydrophilic). As a result, hydrophobic molecules in the polar mobile phase tend to adsorb to the hydrophobic stationary phase, and hydrophilic molecules in the mobile phase will pass through the column and are eluted first. Reversed-phase chromatography is the most common HPLC separation technique and is used for separating compounds that have hydrophobic moieties and do not have a dominant polar character (although polarity of a compound does not exclude the use of RP-HPLC). HPLC stationary phases can be segregated by their ability to separate either polar on nonpolar compounds, that is, reversed-phase materials (C18, C8) strongly retain nonpolar solutes with polar solutes eluting at or near the void volume, and hydrophilic interaction. Silica gel is a polar adsorbent. This allows it to preferentially adsorb other polar materials. When it comes to polarity, materials interact more with like materials. This principle is particularly important to many laboratories, which use silica gel as the stationary phase for column chromatography separations.
Introduction
Published in T. R. Crompton, Determination of Metals and Anions in Soils, Sediments and Sludges, 2020
The most commonly used chromatographic mode used in high-performance liquid chromatography is reversed-phase chromatography. Most common reversed phase chromatography is performed using bonded silica-based columns, thus inherently limiting the operating pH range to 2.0—7.5. High sensitivity detection of non-chromophoric ions can be achieved by combining the power of suppressed conductivity detection with these columns and this is usually a superior approach to using refractive index or low ultra-violet wavelength detection. Often detectors that have been used include rapid diode array detectors and electrochemical detectors. Various companies supply equipment for high-performance liquid chromatography including Dionex, Perkin Elmer, Kontrol, Shimadzu, LKB, Cecil Instruments, Vorian, Isio, Hewlett—Packard, Applied Chromatography Systems, Roth Scientific, PSA Inc (see Appendix 1 for further details).
Instrumentation for High-Performance Liquid Chromatography
Published in Grinberg Nelu, Rodriguez Sonia, Ewing’s Analytical Instrumentation Handbook, Fourth Edition, 2019
Chromatography owes its universality to several separation mechanisms made possible by a variety of stationary and mobile phases. A general survey of the different separation mechanisms available is given in Table 22.1. Separation is based on hydrophobic, polar, ionic molecular, and steric interactions, which selectively enhance or inhibit the partitioning of different analytes into the stationary phase. Columns are often dev eloped to maximize one type of interaction and to minimize all others, although mixed mode columns have become popular to address difficult separations. Widespread applicability, rugged columns, ease of method development, and UV compatible mobile phases make reversed phase chromatography the most widely used. For a comprehensive discussion of method development, the reader is referred to specific references on the subject (Snyder, 1997).
Bioprocessing of recombinant proteins from Escherichia coli inclusion bodies: insights from structure-function relationship for novel applications
Published in Preparative Biochemistry & Biotechnology, 2023
Kajal Kachhawaha, Santanu Singh, Khyati Joshi, Priyanka Nain, Sumit K. Singh
The separation in reversed phase chromatography is based on the differences in the hydrophobicity of various species in a sample. Hence, the method is capable of separating the product related impurities such as oxidized and reduced species formed during protein refolding.[163] However, since RP-HPLC is carried out under denaturing conditions, no information is obtained about the secondary and tertiary structure of the protein. Therefore, the information obtained is merely chemical in nature. Further, the high column temperature used during RP-HPLC might induce protein aggregation. Nevertheless, RP-HPLC being very fast, robust, and high-resolution method is widely used to assess the product homogeneity by measuring the levels of various product-related impurities.[151]
Analytical progress and challenges for the detection of oxygenated polycyclic aromatic hydrocarbon transformation products in aqueous and soil environmental matrices: A review
Published in Critical Reviews in Environmental Science and Technology, 2019
Coren Pulleyblank, Sabrina Cipullo, Pablo Campo, Brian Kelleher, Frederic Coulon
Liquid chromatographic techniques take advantage of the same differences in size, polarity, and acidity that can make addressing the wide array of PAH transformation products difficult. Typically, samples are separated by reverse phase chromatography with C18 used most often as the stationary phase. Although C18 may provide inadequate resolution of the more polar PAH metabolites in complex samples, it has been successfully applied for the separation of diverse oxygenated PAH with different functionalities during the same run, for example, both conjugated and unconjugated metabolites (Malmquist et al., 2013; Tang et al., 2016). Phenyl-modified silica has been preferred by some workers since the enhanced polarizability of the stationary phase can facilitate interaction with aromatic nuclei and improve separation of closely-eluting compounds; methods are available which address up to 81 PAH and oxygenated PAH of varying and mixed functionalities including low molecular weight phenolic acids in the same run (Letzel et al., 2001). Nevertheless, sample complexity and matrix interferences may present significant challenges to achieving adequate separation or identification of analytes, and most workers focus only on a subclass of these compounds after prior fractionation utilizing SPE, online SPE (Olmos-Espejel et al., 2012) or a more selective extraction procedure (Wang et al., 2012).