Name: hplc solvent flow and elution types
Uploaded: 2024-12-05T18:32:00.000Z
Duration: 1 min 5 s
Channel: JoVE

In an HPLC instrument, multiple solvents are stored in solvent reservoirs that facilitate the variation of mobile phase composition and, hence, its polarity.
First, the solvent is filtered and degassed to remove dissolved gases, before it is pumped.
Among high-pressure pumps used in HPLC, the reciprocating pump is the most widely used.
This pump is based on a small piston with two one-way check valves.
When the piston is driven back, the solvent flows in through the inlet valve and is pumped out through an outlet valve during the forward stroke.&#160;
The mobile phase composition is constant in isocratic elution, whereas in gradient elution, it is varied to facilitate separation.
Here, independent binary pumps connected to a mixing chamber regulate the flow rate and polarity of the mobile phase.
Finally, the sample is injected into the flowing mobile phase through a loop injector before it enters the column.

high-performance liquid chromatography: elution process

High-Performance Liquid Chromatography: Elution Process

In High-Performance Liquid Chromatography (HPLC), the elution process is critical to the separation of analytes and the quality of chromatographic results. Elution describes how compounds move through the column and separate based on their interactions with the mobile and stationary phases. This process determines the resolution, peak shape, and retention times in the chromatogram, which are essential for identifying and quantifying components in complex mixtures. Understanding the elution process helps in interpreting chromatographic data and improving separation efficiency.
HPLC utilizes highly pure HPLC-grade solvents to ensure minimal interference from impurities. High-grade solvents are essential because even trace amounts of contaminants can interfere with the elution process, distort chromatogram peaks, and reduce sensitivity during sample detection. Moreover, solvents may contain dissolved gases such as nitrogen and oxygen, which can also interfere with sample detection. Degassing is employed to eliminate these gases, using either a vacuum pump or purging with Helium gas, to maintain the integrity of the separation and detection processes.
The degassed solvents are then pulled from the mobile phase reservoir by a high-pressure pump, with the reciprocating pump being the most commonly used type. The pump consists of a piston and two check valves, which draw the solvent using the back-and-forth movement of the piston. However, a disadvantage of the reciprocating pump is that it produces a pulsed solvent flow. Therefore, modern HPLC systems have a pulse damper connected to the pump to address this issue. Other pumps, such as screw-driven displacement pumps, are available but may have limitations such as less solvent capacity and low-pressure bearing capacity, which make them less convenient for solvent changes.
There are two types of elution processes: isocratic and gradient elution. Isocratic elution uses a single solvent or mobile phase throughout the separation, while gradient elution involves a change in the composition during the separation process. This method improves separation efficiency, reduces analysis time, and enhances resolution, leading to sharper peaks in the chromatogram. Gradient elution provides better resolution and results in more distinct and separated peaks, making it easier to interpret the chromatogram for complex mixtures compared to isocratic elution.
Before the mobile phase enters the column, the sample is introduced using a loop injector with a two-position valve. The valve helps to direct the mobile phase to the column. Due to the high pressure in HPLC, sample injection cannot be performed as in gas chromatography (GC). Sampling loops, with fixed volumes ranging from 0.5 microliters to 2 milliliters, can be interchangeable.

In High-Performance Liquid Chromatography (HPLC), the elution process is critical to the separation of analytes and the quality of chromatographic ...

high-performance-liquid-chromatography-elution-process

Education

JoVE Core

Analytical Chemistry

Unit 1

Principles of Chromatography

KEY TERMS AND CONCEPTS

hplc solvent flow and elution types

High-performance liquid chromatography (HPLC) uses solvent flow and elution to separate analytes in a column. Elution is the way compounds move through the column and separate because of how they interact with the mobile phase and the stationary phase. These interactions affect retention times, peak shape, and resolution in the chromatogram. Knowing how elution works helps students read chromatographic data and understand how mixtures are separated.

HPLC relies on highly pure HPLC-grade solvents so that impurities do not disturb the separation. Even small amounts of contaminants can distort chromatogram peaks and lower detection sensitivity. The solvents can also contain dissolved gases such as nitrogen and oxygen. To protect the analysis, the solvents are degassed first, either with a vacuum pump or by purging with helium gas.

After degassing, the mobile phase is drawn from the reservoir by a high-pressure pump. The most common type is the reciprocating pump, which uses a piston and two check valves to pull solvent with a back-and-forth motion. One drawback is that it creates a pulsed solvent flow. Modern HPLC systems often add a pulse damper to smooth that flow, although screw-driven displacement pumps are also used and may have limits such as lower solvent capacity and lower pressure-bearing ability.

HPLC can run with isocratic or gradient elution. Isocratic elution uses one solvent or mobile phase for the entire separation. Gradient elution changes the solvent composition during the run, which can improve separation efficiency, shorten analysis time, and sharpen peaks. This makes the chromatogram easier to interpret, especially for complex mixtures, because the peaks become more distinct.

Before the mobile phase enters the column, the sample is added with a loop injector and a two-position valve. The valve directs the mobile phase to the column, which is important because HPLC runs under high pressure. Sample injection cannot be done the same way as in gas chromatography (GC). Sampling loops have fixed volumes, and interchangeable loops can range from 0.5 microliters to 2 milliliters.

HPLC Solvent Flow and Elution Types

chromatographic methods: terminology

Chromatography Terms and Peak Analysis

Chromatography is an analytical technique used to separate the parts of a mixture and identify substances in it. It is used in chemistry, biology, environmental science, and pharmaceuticals. The method depends on two phases: the stationary phase, which stays fixed, and the mobile phase, which moves across it and carries the solutes. As the mobile phase travels, each mixture component interacts with the phases in a different way. That difference causes the components to separate. Chromatography...

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Chromatography Types and Separation Modes

Chromatographic techniques are grouped by the phases they use, how those phases interact, and the way they separate sample components. The mobile phase is usually a liquid or a gas. The stationary phase is usually a solid or a liquid layer on a solid surface. Many chromatography names start with the type of chromatography, then the mobile phase, and then the stationary phase. For example, gas-solid chromatography uses a gas mobile phase and a solid stationary phase. In liquid-liquid...

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Chromatography separates solutes by how they move between the mobile phase and the stationary phase. In many systems, this transfer happens through sorption, which usually means adsorption. The separation depends strongly on polarity, or the overall dipole moment of a molecule. During chromatography, the solute and the solvent compete for adsorption on the stationary phase. Highly polar compounds and polar solvents tend to adsorb strongly on polar surfaces. This affects how the mixture...

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Band Broadening from Column Diffusion

Column diffusion causes band broadening in chromatography columns. When an analyte is added as a narrow band at the top of the column, the solutes begin to separate and spread out. As this happens, the band develops a Gaussian profile, which means the concentration is highest near the center and lower at the edges. Longitudinal diffusion is one reason this spreading occurs. In this process, solute molecules in the mobile phase move from the more concentrated center of the band into the more...

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Peak Separation in Chromatography

Peak separation in chromatography depends on how far apart two neighboring peaks appear in a chromatogram. As a solute moves through a chromatographic column, it spreads into a band with a Gaussian shape. The longer the solute stays in the column, the broader that band becomes. This broadening can cause peaks to overlap and reduce separation efficiency. Chromatographic resolution is the measure used to judge that separation. It is the ratio of the difference in retention time or retention...

column efficiency: plate theory

Theoretical Plates in Chromatography Columns

Theoretical plates help explain how chromatography columns achieve good separation. Column efficiency is measured by band broadening, and that efficiency depends on the number of theoretical plates, or N. In plate theory, a column is treated as a continuous series of imaginary plates where solute equilibration happens between the stationary and mobile phases. A higher number of theoretical plates means better column efficiency and stronger separation ability. Plate height affects both band...

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Chromatography Band Broadening in Columns

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optimizing chromatographic separations

Improving Chromatography Resolution

Chromatography resolution improves when bands stay narrow and the two solutes separate clearly in the column. Good separation often depends on band broadening, plate height, capacity factor, and separation factor. These ideas help explain why some mixtures come apart quickly while others take more time. Band broadening is the spreading of solute bands as they move through the column. When bands spread out, resolution drops. Plate height, or H, is the length needed for one theoretical plate. A...

silica gel column chromatography: overview

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Silica Gel Column Chromatography and Polarity

Silica gel column chromatography separates compounds by polarity using a column packed with silica gel. The silica gel acts as the stationary phase, and the solvent is the mobile phase. As the mixture moves through the column, the compounds separate into distinct bands. Polar compounds tend to bind more strongly to the silica gel. Because of this stronger interaction, they move more slowly through the column. Nonpolar compounds bind more weakly to the silica gel. They usually travel faster...

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TLC Separation by Polarity

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Gas Chromatography Detector Types and Uses

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Specialized Detectors in Gas Chromatography

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high-performance liquid chromatography: introduction

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HPLC Separation, Columns, and Detection

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High-performance liquid chromatography (HPLC) uses solvent flow and elution to separate analytes in a column. Elution is the way compounds move through the column and separate because of how they interact with the mobile phase and the stationary phase. These interactions affect retention times, peak shape, and resolution in the chromatogram. Knowing how elution works helps students read chromatographic data and understand how mixtures are separated. HPLC relies on highly pure HPLC-grade...

high-performance liquid chromatography: instrumentation

high-performance-liquid-chromatography-instrumentation

HPLC Parts and How They Work

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high-performance liquid chromatography: types of detectors

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HPLC Detector Choices for Analyzing Solutes

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Size-Exclusion Chromatography in Protein Analysis

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capillary electrophoresis: instrumentation

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Capillary Electrophoresis: Sample Separation Setup

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key terms and concepts

Learn how HPLC solvent flow, degassing, pumps, and isocratic vs. gradient elution affect separation, peak shape, and chromatogram quality.

HPLC Solvent Flow and Elution Types | JoVE K12

analytical chemistry

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HPLC Solvent Flow and Elution Types

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