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Current Perspectives and Methods for the Characterization of Natural Medicines
Published in Rohit Dutt, Anil K. Sharma, Raj K. Keservani, Vandana Garg, Promising Drug Molecules of Natural Origin, 2020
Muthusamy Ramesh, Arunachalam Muthuraman, Nallapilai Paramakrishnan, Balasubramanyam I. Vishwanathan
High-performance liquid chromatography is also called HPLC. It is an advanced analytical technique over column chromatography. In this technique, the mobile phase is pumped with pressure through a stationary phase of the column which is packed irregularly shaped particles to speed up the analytical procedure. The solvent phase is pumped through a column with a pressure of, 1000–3000 psi. HPLC is effective techniques in the analysis of phytochemical and marine constituents. The approach speeds up the analysis over the traditional column. HPLC is carry small volume of liquid as a sample is injected into the column comprising porous particles (stationary phase). The particle size varies from 5–10 µM. When mixtures of compounds are passed into the column, components are isolated based on their interaction property between the sample and stationary phase by the movement of liquid. The separated components are detected by part of the HPLC device is called detector. Generally HPLC has two method of operation, based on their working principle employed on separation i) normal phase chromatography; and ii) reverse phase chromatography. Normally, chromatography techniques are working based on polar (hydrophilic) stationary phase and non-polar (hydrophobic)mobile phase. In HPLC, it is reverse phase functions, i.e., non-polar stationary phase and polar mobile phase. As many of the pharmaceuticals/drugs are polar in nature, reverse phase HPLC is mainly used in pharmaceutical industries. Based on the principle employed on elution, HPLC is classified into two different categories: (i) isocratic elution; and (ii) gradient elution. The polar component of the mobile phase is constant for isocratic elution. In contrast, the gradient elution pattern, the mobile phase is the reversed process of HPLC analysis. Gradient elution is reduced the retention time and therefore, the components are eluted faster. The approach improves the shape and height of the peak. Further, HPLC is classified into two different modes of operation: (i) analytical HPLC (the compounds are not recovered; (ii) preparative HPLC (the compounds are recovered). The instrumentation of HPLC includes a solvent reservoir, mixing vessel, pressure pump, guard column, a sample injector, column, detector, and collector. Retention time, retention volume, separation factor, resolution, height equivalent theoretical plate, efficiency, and asymmetry factor are the HPLC are employed to detects the components from the test sample. A schematic diagram of the instrumentation of high-performance liquid chromatography is illustrated in Figure 2.5.
Mass spectrometry-based metabolomics diagnostics – myth or reality?
Published in Expert Review of Proteomics, 2021
Oxana P. Trifonova, Dmitri L. Maslov, Elena E. Balashova, Petr G. Lokhov
Despite the promising results, there are numerous technical and biological challenges in MS-based metabolomics diagnostics. Using various MS-based techniques in metabolomics enables detection and quantification (absolute or relative) of hundreds of distinct metabolites in complex biological samples. However, each of these technologies has its advantages and limitations [3,44]. For example, gas chromatography-mass spectrometry (GC–MS) is considered as one of the most suitable and inexpensive techniques for metabolite analysis due to its high resolution and selectivity, excellent separation efficiency and reproducibility, and easy component identification using universal commercial and public databases. However, for analysis, sample derivatization is required and therefore, only volatile metabolites (or those that can be volatilized (e.g. most amino acids, sugar alcohols, aromatic amines, and organic acids)) can be analyzed [45,46]. Liquid chromatography-MS (LC-MS) methods are characterized by excellent sensitivity and resolution, simple sample preparation, and the ability to analyze metabolites from different classes. However, matrix effect due to ion suppression and difficulties with metabolite identification also exist. Using different chromatographic columns, the LC-MS method has a wider metabolite coverage and allows the analysis of both nonpolar (reversed-phase chromatography) and polar (normal phase chromatography) compounds [47,48]. In addition to the popular LC-MS and GC-MS techniques, there are also direct-injection MS (DIMS) [27,49] and capillary electrophoresis-mass spectrometry (CE-MS) [50,51] methods, which also have a significant impact on MS-based metabolomics diagnostics. Indeed, there is no universal analytical platform for measuring the entire metabolome because of the wide ranges in concentration (from g/L to ng/L and lower), and diversity in physicochemical properties of metabolites. This makes analysis of the complete metabolome technically challenging [2].
Macrocarpal C isolated from Eucalyptus globulus inhibits dipeptidyl peptidase 4 in an aggregated form
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2018
Eisuke Kato, Kazuhiro Kawakami, Jun Kawabata
E. globulus extract was selected for further study and separated using an activity-guided procedure. Repeated separation by normal phase chromatography and reverse phase HPLC yielded macrocarpals A, B and C (Figure 1), which were identified by NMR and MS analyses, with reference to published data9,10,12.
Human disease glycomics: technology advances enabling protein glycosylation analysis – part 1
Published in Expert Review of Proteomics, 2018
Arun V Everest-Dass, Edward S X Moh, Christopher Ashwood, Abdulrahman M M Shathili, Nicolle H Packer
HILIC offers separation on the basis of glycan hydrophilicity, that is, dependent on properties such as size, charge, composition, structure, and linkage. HILIC is normal-phase chromatography, where an aqueous phase is usually used as the eluting solvent with an amide-derivatized silica as stationary phase.