Reversed Phase Chromatography
Reversed Phase Chromatography - Related Videos
03:31
Reversed-Phase High-Performance Liquid Chromatography: A Robust Method for Quantitation of Fluorescently Labeled and Derivatized Sialic Acids Isolated from Mouse Liver
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2025
In this video, we demonstrate the detection of sialic acids, N-acetylneuraminic acid and N-glycolylneuraminic acid, using high-performance liquid chromatography, HPLC, following derivatization with a suitable fluorescent labeling reagent to aid in fluorescence detection.
08:06
Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry
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Cited by 16 •
2019
Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.
10:14
Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography
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Cited by 3 •
2014
A protocol for the diastereoselective one-pot preparation of cis-N-Ts-iodoaziridines is described. The generation of diiodomethyllithium, addition to N-Ts aldimines and cyclization of the amino gem-diiodide intermediate to iodoaziridines is demonstrated. Also included is a protocol to rapidly and quantitatively assess the most appropriate stationary phase for purification by chromatography.
09:23
Column Chromatography
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2023
Source: Laboratory of Dr. Jimmy Franco - Merrimack College Column chromatography is one of the most useful techniques for purifying compounds. This technique utilizes a stationary phase, which is packed in a column, and a mobile phase that passes through the column. This technique exploits the differences in polarity between compounds, allowing the molecules to be facilely separated.1 The two most common stationary phases for column chromatography are silica gel (SiO2) and alumina (Al2O3), with...
01:17
Gas Chromatography: Types of Columns and Stationary Phases
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2024
Gas chromatography (GC) relies on stationary phases to separate and analyze components in a sample. There are two main types of stationary phases: liquid and solid. Liquid stationary phases are non-volatile, thermally stable, and chemically inert liquids coated onto the column. Solid stationary phases are particles of adsorbent material, such as silica gel or molecular sieves. For an analyte to remain on the column for a sufficient amount of time, it must exhibit some level of compatibility (or...
FAQs
Retention is governed by the relative preference of each compound for the nonpolar stationary phase versus the polar mobile phase. Compounds that interact more strongly with the alkyl-modified surface spend longer in the column and elute later, while compounds with weaker interactions emerge earlier. This ordering provides a basis for comparing mixture components and assessing separation.
Solvent composition changes the balance between retention and movement through the column. A water-organic solvent mixture can therefore be adjusted to alter how strongly mixture components remain associated with the nonpolar surface. Researchers use these changes to tune elution behavior, improve resolution between compounds, and make separations more suitable for identification, purification, or quantification.
Optimization is necessary because pH, temperature, and flow rate can each influence retention and resolution. Rather than treating these settings as fixed, researchers adjust them to obtain a more useful separation for the particular mixture. The goal is controlled elution with enough distinction among components to support reliable identification, purification, or quantification.
A reversed phase chromatography setup combines a nonpolar stationary phase with a more polar mobile phase. The stationary material commonly consists of silica modified with alkyl groups, while the mobile phase often contains water and an organic solvent. Choosing and balancing these components establishes the chemical environment that governs how mixture constituents are retained and separated.
A practical workflow starts by selecting the modified-silica stationary phase and water-organic mobile phase, then setting pH, temperature, and flow rate for the mixture. After the sample is separated, the resulting elution behavior can be used to identify components, isolate desired materials for purification, or measure their amounts. Condition changes are made when resolution is insufficient.
Its broad utility comes from combining separation with information about compound behavior in complex mixtures. Chemists apply it to pharmaceuticals, biomolecules, environmental samples, and other chemically diverse materials. Depending on the objective, the separation can support component identification, purification of selected substances, or quantitative analysis of how much of a compound is present.