Tag Archives: chromatography

Ion-Exchange Chromatography

In ion-exchange chromatography (IEC) the stationary phase is a cross-linked polymer resin, usually divinylbenzene cross-linked polystyrene, with covalently attached ionic functional groups. As shown here for a styrene–divinylbenzene co-polymer modified for use as an ion-exchange resin, the ion-exchange sites—indicated here by … Continue reading

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LC-MS Detection

The figure below provides a block diagram of a typical liquid chromatography-mass spectrometer (LS–MS) instrument. As illustrated here, a three component mixture is injected onto the HPLC. When component A elutes from the column, it enters the MS ion source and ionizes … Continue reading

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Electrochemical Detector for HPLC Separations

Another common group of HPLC detectors are based on electrochemical measurements such as amperometry, voltammetry, coulometry, and conductivity. The illustration below, for example, shows an amperometric flow cell. Effluent from the column passes over the working electrode, which is held … Continue reading

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Absorbance and Fluorescence Detection for HPLC Separations

One popular HPLC detector takes advantage of an analyte’s UV/Vis absorption spectrum. These detectors range from simple designs, in which the analytical wavelength is selected using appropriate filters, to a modified spectrophotometer in which the sample compartment includes a flow … Continue reading

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Loop Injector for HPLC

The operating pressure of an HPLC is sufficiently high that we cannot inject the sample into the mobile phase by inserting a syringe through a septum (as we can do in gas chromatography). Instead, we inject the sample using a … Continue reading

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HPLC Pumps

A typical HPLC includes between 1–4 reservoirs for mobile phase solvents. The mobile phase solvents are pulled from their reservoirs by the action of one or more pumps. Shown here is a close-up view of an instrument with two pumps, each … Continue reading

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Gradient Elution Separation of Flavonoid Mixture

The illustration below shows the gradient elution separation of a mixture of flavonoids. Mobile phase A is an aqueous solution of 0.1% formic acid and mobile phase B is 0.1% formic acid in acetonitrile. The initial mobile phase is 98% … Continue reading

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Using a Solvent Triangle to Optimize an HPLC Separation

One strategy for finding the best mobile phase for an HPLC separation is to use the solvent triangle, which allows us to explore a broad range of mobile phases with just seven experiments. We begin by adjusting the amount of acetonitrile … Continue reading

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HPLC Column

The most common type of HPLC column is a stainless steel tube with an internal diameter between 2.1 mm and 4.6 mm and a length between 30 mm and 300 mm. The column is packed with 3–10 μm porous silica particles … Continue reading

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HPLC Instrumentation

The photo below shows an example of a typical HPLC instrument with insets showing close-up views of the pumps that move the mobile phase through the system, and the plumbing used to inject the sample into the mobile phase. This particular instrument … Continue reading

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