HPLC Chromatography | Animation | High Performance Liquid Chromatography | Remake

Dr. Puspendra Classes
15 Feb 202105:18
EducationalLearning
32 Likes 10 Comments

TLDRThis animation delves into the intricacies of High Performance Liquid Chromatography (HPLC), detailing its components from solvent reservoirs to detectors and recorders. It explains the process, starting from the preparation of the mobile phase, through the sample injection, to the separation of compounds in the analytical column based on their affinity with the stationary phase. The video aims to educate viewers on the HPLC technique and encourages sharing for educational purposes, with a request for permission for commercial use.

Takeaways
  • 🌟 HPLC stands for High Performance Liquid Chromatography, a technique used for the separation of compounds in a mixture.
  • 💧 The HPLC system includes various components such as a solvent reservoir, pump, degasser, sample injector, pre-column, analytical column, detector, and recorder.
  • 🔁 The solvent reservoir holds different solvents which are pumped through the system by the HPLC pump.
  • 🌀 The degasser, connected to a vacuum pump, removes dissolved gases from the solvents to prevent issues during chromatography.
  • 🔄 The solvent mixing valve ensures a uniform mobile phase by mixing the solvents from the reservoir.
  • 🛡️ The pre-column, also known as the guard column, protects the analytical column from impurities and removes any contaminants from the mobile phase.
  • 🧪 The analytical column, typically made of stainless steel and filled with a stationary phase, is where the actual separation of compounds occurs.
  • 🔍 The detector measures the separated compounds as they exit the analytical column, providing signals that correspond to the compounds' properties.
  • 📊 The recorder captures the signals from the detector, creating a chromatogram that visually represents the separation process.
  • 📝 The HPLC method involves setting parameters such as solvent ratio, flow rate, runtime, and type of elution using the HPLC software.
  • 🔑 The stationary phase commonly used in HPLC is octadecyl silin silica gel, also known as ODS, and the columns are often referred to as C18 columns.
  • 📚 The script suggests sharing the educational content for non-commercial purposes and obtaining permission for any commercial use.
Q & A

  • What is the full form of HPLC?

    -The full form of HPLC is High Performance Liquid Chromatography.

  • What are the main components of an HPLC instrument?

    -The main components of an HPLC instrument include a solvent reservoir, HPLC pump with degasser, solvent mixing valve, sample injector, pre-column (guard column), analytical column, detector, and recorder.

  • What is the purpose of the degasser in an HPLC system?

    -The degasser is used to remove any dissolved gases or air from the solvents to prevent issues during the elution process.

  • What is the function of the pre-column or guard column in HPLC?

    -The pre-column or guard column serves to remove impurities from the mobile phase and protect the analytical column from contamination.

  • What is the typical length and internal diameter of a pre-column in HPLC?

    -The typical length of a pre-column is between 2 to 10 centimeters, and the internal diameter is usually 4.6 millimeters.

  • What is the common stationary phase used in HPLC columns?

    -The commonly used stationary phase in HPLC columns is octadecyl cyclin silica gel, also known as ODS silica gel.

  • Why is it important to condition the HPLC instrument with blank mobile phase before sample injection?

    -Conditioning the HPLC instrument with blank mobile phase for at least 10 minutes ensures proper setup and prevents any initial anomalies in the chromatographic run.

  • How does the sample get injected into the HPLC system?

    -The sample is injected into the HPLC system through a sample injector using a micro syringe.

  • What causes the separation of compounds in the analytical column of an HPLC?

    -The separation of compounds in the analytical column is due to the differences in their affinity with the stationary phase.

  • How does the detector in an HPLC system work?

    -The detector in an HPLC system detects the separated compounds as they elute from the column and gives signals that are recorded, which can be analyzed to identify and quantify the compounds.

  • What is the significance of the color coding in the script's description of the detector signals?

    -The color coding (blue, red, green) is used to illustrate the order in which compounds with varying affinities with the stationary phase reach the detector, with blue indicating the first to be detected and green the last.

  • Can the provided animation be used for commercial purposes without permission?

    -No, the animation cannot be used for commercial purposes without obtaining written permission from the creators through the provided email ID.

Outlines
00:00
🔬 Introduction to HPLC Instrumentation and Operation

This paragraph introduces the concept of High Performance Liquid Chromatography (HPLC), detailing the components of an HPLC system including the solvent reservoir, pump, degasser, sample injector, pre-column, analytical column, detector, and recorder. It explains the materials used for the columns and the stationary phase, specifically mentioning octadecyl silin silica gel or ODS. The paragraph also outlines the process of setting up an HPLC method, degassing the solvent, mixing the mobile phase, and the role of the guard column in protecting the analytical column from impurities. The summary concludes with the injection of the sample and its journey through the system, leading to the separation of compounds based on their affinity to the stationary phase and their detection by the detector.

05:01
📝 Usage Permissions and Acknowledgments

The second paragraph discusses the conditions for using the provided HPLC animation. It emphasizes the need for written permission for any commercial use of the material, with the contact information provided on-screen. The speaker thanks the audience and encourages them to share the video to help cover production costs and enable the creation of more educational content. The paragraph also suggests that teachers can use the animation for non-commercial educational purposes to explain HPLC to their students.

Mindmap
Keywords
💡HPLC
HPLC stands for High Performance Liquid Chromatography, a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It is central to the video's theme, as the script provides an overview of its instrumentation and working principles. The script describes the components of an HPLC system and how they work together to perform chromatographic separations.
💡Solvent Reservoir
A solvent reservoir is a container that holds the liquid solvents used in HPLC. It is essential for the HPLC system as it supplies the mobile phase, which carries the sample through the chromatographic process. The script mentions that the HPLC pump draws solvent from the reservoir towards the degasser.
💡Degasser
A degasser is a device attached to an HPLC system that removes dissolved gases from the mobile phase to prevent air bubbles, which can interfere with the separation process. The script explains that the degasser is connected to a vacuum pump to facilitate this removal process.
💡Solvent Mixing Valve
The solvent mixing valve is a component that allows for the mixing of different solvents to create a uniform mobile phase. It is crucial for ensuring consistent conditions throughout the HPLC run, as mentioned in the script where it states that mixing occurs to produce a uniform mobile phase.
💡Sample Injector
The sample injector is the part of the HPLC system where the sample is introduced into the mobile phase. It is vital for starting the separation process, as highlighted in the script where it describes the injection of the sample using a micro syringe.
💡Pre-column (Guard Column)
A pre-column, also known as a guard column, is a short column placed before the analytical column to protect it from impurities in the sample or mobile phase. The script specifies its function and dimensions, emphasizing its role in safeguarding the analytical column.
💡Analytical Column
The analytical column is the main component of the HPLC system where the separation of compounds occurs. It has a specific length and internal diameter, and is filled with a stationary phase. The script describes its dimensions and the materials used for the stationary phase.
💡Stationary Phase
The stationary phase is the material within the HPLC column that interacts with the compounds in the sample, leading to their separation. The script mentions octadecyl cylin silica gel, also known as ODS silica gel, as a common stationary phase, and refers to the columns containing this as C18 columns.
💡Detector
The detector in an HPLC system is responsible for identifying and quantifying the separated compounds as they exit the analytical column. It provides signals that are then recorded, as described in the script where it explains how compounds with different affinities produce different signals.
💡Recorder
The recorder is the component that records the output from the detector, creating a chromatogram that visually represents the separation of compounds. The script mentions the recorder as the final part of the HPLC process where all signals are captured.
💡Affinity
Affinity in the context of HPLC refers to the strength of interaction between the compounds in the sample and the stationary phase. The script uses the term to explain how compounds with varying affinities are separated, with those having the least affinity reaching the detector first.
Highlights

Introduction to HPLC and its full form.

Description of the HPLC instrument's construction from various parts.

Explanation of the solvent reservoir and its role in HPLC.

Functioning of the HPLC pump and degasser with a vacuum pump.

Introduction to the solvent mixing valve and sample injector.

Details about the pre-column, also known as the guard column.

Specifications of the guard column's length and internal diameter.

Introduction to the analytical column and its dimensions.

Composition of the analytical and pre-columns made of stainless steel.

Use of stationary phase in HPLC columns, specifically ODS silica gel.

Common naming convention for HPLC columns, such as C18.

Role of the detector in the HPLC process.

Functioning of the recorder in capturing HPLC data.

Process of starting the HPLC instrument and entering parameters.

Pumping of the mobile phase from the solvent reservoir.

Degassing process to remove dissolved gases from solvents.

Mixing of solvents to produce a uniform mobile phase.

Role of the guard column in removing impurities and protecting the analytical column.

Description of the sample injection process using a micro syringe.

Mechanism of sample separation in the analytical column based on affinity differences.

Signal generation in the detector and its representation in colors.

Final appearance of signals in the recorder for analysis.

Call to action for sharing the video for educational purposes.

Request for written permission for commercial use of the animation.

Transcripts

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HPLCInstrumentationChromatographyAnalytical ChemistrySolvent ReservoirDegasserSample InjectorGuard ColumnAnalytical ColumnStationary PhaseDetector