HPLC chromatography

Shomu's Biology

4 Jul 201616:28

Summary

TLDRThe transcript explains High-Performance Liquid Chromatography (HPLC), a technique used in various fields like pharmaceuticals and forensics to separate and identify compounds in a liquid sample. HPLC operates by passing a sample through a column with a stationary phase, using a high-pressure pump and a mobile phase solvent. The retention time varies based on the interaction between the phases and analytes. The summary also touches on different HPLC types, including Ultra High-Performance Liquid Chromatography (uHPLC) and Fast Protein Liquid Chromatography (FPLC), highlighting their specific features and applications.

Takeaways

🔬 High Performance Liquid Chromatography (HPLC) is a technique used to separate, identify, and quantify compounds in a sample mixture by pumping it through a column with a stationary phase under high pressure.
💧 The sample is carried by a moving carrier gas, typically helium or nitrogen, through the column, and interactions between the stationary phase and the mobile phase determine the separation of analytes.
⚗️ HPLC is versatile and can detect compounds at trace concentrations as low as parts per trillion, making it valuable in pharmaceutical, environmental, forensic, and chemical applications.
🧪 The retention time of a sample in HPLC depends on the interaction between the stationary phase, the analytes, and the solvent used in the mobile phase.
🚰 Main components of an HPLC system include solvent reservoirs, a high-pressure pump, a column, an injector system, and a detector.
🖥️ Modern HPLC systems are often fully automated and computer-controlled, allowing for more efficient and precise analysis.
🔍 A detector is used to observe the separated compound bands as they exit the column, and the resulting data is used to generate a chromatogram.
🎈 Helium sparging is commonly used to degas the mobile phase, while nitrogen is used in Evaporative Light Scattering Detectors (ELSD) to measure evaporated solvents.
⚡ Ultra High Performance Liquid Chromatography (uHPLC) uses smaller column particles and higher pressures for faster and more sensitive analysis, but the columns may degrade quicker under high pressure.
🧬 Fast Protein Liquid Chromatography (FPLC) is a similar system to HPLC but is specifically used for separating proteins and biomolecules, with lower pressure capabilities.

Q & A

What is High Performance Liquid Chromatography (HPLC)?
-HPLC is a form of column chromatography that pumps a sample mixture or analyte in a solvent (mobile phase) at high pressure through a column with chromatographic packing material (stationary phase) to separate and identify compounds present in a sample.
What are the primary applications of HPLC?
-HPLC is used in a variety of industrial and scientific applications, including pharmaceutical analysis, environmental testing, forensic science, and chemical research.
How does sample retention time vary in HPLC?
-Sample retention time varies depending on the interaction between the stationary phase, the molecules being analyzed, and the solvent(s) used. Analytes with less interaction with the stationary phase or more interaction with the mobile phase exit the column faster.
What are the main components of an HPLC system?
-The main components of an HPLC system include the solvent reservoir, high-pressure pump, column, injector system, and detector. The system is usually automated and controlled by a computer.
What role does the solvent reservoir play in HPLC?
-The solvent reservoir holds the solvent, known as the mobile phase, which moves through the system. Typically, there are at least two reservoirs in a system, each with up to 1000 cc of solvent.
Why is helium sparging used in HPLC?
-Helium sparging is used to degas the mobile phase, which helps to avoid unstable baselines caused by dissolved air in the solvent.
What is Ultra High Performance Liquid Chromatography (uHPLC), and how does it differ from standard HPLC?
-uHPLC is a more advanced version of HPLC that uses smaller column particles (down to 1.7μm) and operates at higher pressures (exceeding 1000 bar). It is faster, more sensitive, and uses smaller volumes of solvents, but the columns degrade quicker under higher pressures.
What is Fast Protein Liquid Chromatography (FPLC), and how does it compare to HPLC?
-FPLC is a chromatography technique similar to HPLC, used to separate or purify proteins and other biomolecules. The main difference is that FPLC operates at lower pressures (3-5 MPa), making it suitable for biomolecules that might be damaged at the higher pressures used in HPLC.
Can FPLC columns be used in HPLC systems?
-Yes, FPLC columns can be used in HPLC systems if the pressure is limited to the levels suitable for FPLC, typically up to 3-5 MPa.
What is the primary advantage of uHPLC over standard HPLC?
-The primary advantage of uHPLC over standard HPLC is its speed, as it can run more samples in less time due to its higher sensitivity and the use of smaller volumes of organic solvents.

Outlines

00:00

🔬 Introduction to High Performance Liquid Chromatography (HPLC)

High Performance Liquid Chromatography (HPLC) is a type of column chromatography where a sample mixture in a solvent, known as the mobile phase, is pumped at high pressure through a column with a stationary phase. This technique allows for the separation and identification of compounds in samples that can be dissolved in liquid, even at trace concentrations as low as parts per trillion. HPLC is versatile and widely used in industries like pharmaceuticals, environmental science, forensics, and chemical analysis. The retention time of samples varies based on the interaction between the stationary phase, the analyte molecules, and the solvents used. Analytes with less interaction with the stationary phase or more interaction with the mobile phase exit the column faster. An HPLC system includes key components such as the solvent reservoir, high-pressure pump, column, injector system, and detector. The system is often automated and controlled by a computer that generates a chromatogram as the compounds separate. Helium sparging is used to degas the mobile phase to prevent unstable baselines caused by dissolved air.

05:17

🌡️ Advanced HPLC Techniques: uHPLC and ELSD

Ultra High Performance Liquid Chromatography (uHPLC) is an advanced form of HPLC that uses smaller column particles (down to 1.7 µm) and higher pressures (over 1000 bar) compared to standard HPLC. The primary advantage of uHPLC is its speed, allowing for faster analysis, greater sensitivity, and reduced solvent use, leading to more efficient sample processing. However, operating at pressures above 800 bar can cause faster degradation of columns. Advances in technology are enabling uHPLC systems to use even smaller particles and higher pressures (up to 6,800 bar). uHPLC is also known as UPLC™, a trademark of the Waters Corporation. In addition, Evaporative Light Scattering Detectors (ELSD) use nitrogen as a nebulization gas to evaporate the solvent from the sample, leaving a mist that is measured. These innovations improve HPLC's efficiency and capabilities in analyzing complex mixtures.

10:20

🧪 Fast Protein Liquid Chromatography (FPLC) Explained

Fast Protein Liquid Chromatography (FPLC) is a system similar to HPLC but designed for separating or purifying proteins and other biomolecules from complex mixtures. The primary difference between FPLC and HPLC is the standard working pressure. FPLC columns operate at lower pressures, typically between 3-5 MPa. Despite the lower pressure, FPLC columns can be used in HPLC systems if the pressure is adequately controlled. This flexibility makes FPLC a valuable tool for protein analysis, complementing the capabilities of HPLC in biochemical and molecular biology research.

Mindmap

Keywords

💡High Performance Liquid Chromatography (HPLC)

HPLC is 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 it describes the process and applications of this technique. The script explains that HPLC uses a high-pressure liquid to carry a sample mixture through a column packed with a stationary phase, allowing for the separation of compounds based on their interactions with the phases.

💡Mobile Phase

In HPLC, the mobile phase refers to the solvent that carries the sample mixture through the column. It is key to the chromatographic process, as described in the script, where it interacts with the analytes and affects their rate of movement through the column, thereby influencing the separation of compounds.

💡Stationary Phase

The stationary phase in HPLC is the chromatographic packing material held in place within the column. It is crucial for the separation process, as it provides a surface with which the analytes in the mobile phase can interact. The script mentions that the stationary phase is composed of specific materials needed for the separation of different compounds.

💡Analyte

An analyte is the substance or mixture that is being analyzed in an HPLC process. The script explains that HPLC can separate and identify compounds present in any analyte that can be dissolved in a liquid, highlighting its versatility in various applications.

💡Sample Retention Time

Sample retention time in HPLC is the time it takes for a specific compound to pass through the column. The script describes how this time varies depending on the interaction between the analyte molecules and the stationary and mobile phases, which is a critical parameter in identifying and quantifying compounds.

💡Polarity

Polarity is a property of molecules that affects their interaction with other molecules, which is a significant factor in HPLC separation. The script explains that analytes with different polarities interact differently with the mobile and stationary phases, influencing their movement rate through the column.

💡Injector System

The injector system in HPLC is responsible for introducing the sample into the mobile phase stream. As mentioned in the script, modern HPLC systems are often automated, with auto samplers that facilitate the injection process, ensuring precision and repeatability in sample introduction.

💡Detector

A detector in HPLC is used to monitor the separated compounds as they elute from the column. The script describes how the detector captures the presence of these compounds and sends the information to a computer, which then generates a chromatogram, a graphical representation of the separation process.

💡Chromatogram

A chromatogram is the visual output of an HPLC run, showing the separated compounds over time. The script explains that the computer generates this graph based on the signals from the detector, which is essential for analyzing and interpreting the results of the HPLC process.

💡Degassing

Degassing is the process of removing dissolved gases, such as air, from the mobile phase to prevent instability in the chromatographic baseline. The script mentions helium sparging as an effective method of degassing, which is important for maintaining the reliability of HPLC results.

💡Ultra High Performance Liquid Chromatography (uHPLC)

uHPLC is an advancement of HPLC that operates at higher pressures and uses smaller column particles for faster and more efficient separations. The script describes uHPLC as being faster and more sensitive than standard HPLC, allowing for the analysis of more samples in less time, although it also notes the potential for quicker column degradation at higher pressures.

💡Fast Protein Liquid Chromatography (FPLC)

FPLC is a variation of HPLC specifically designed for the separation and purification of proteins and biomolecules. The script explains that the main difference between FPLC and HPLC is the maximum working pressure, with FPLC operating at lower pressures, making it suitable for certain types of biological samples.

Highlights

High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture in a solvent at high pressure through a column with chromatographic packing material.

HPLC has the ability to separate and identify compounds present in any sample that can be dissolved in a liquid, in trace concentrations as low as parts per trillion.

HPLC is used in various applications such as pharmaceuticals, environmental, forensics, and chemicals due to its versatility.

Sample retention time in HPLC varies depending on the interaction between the stationary phase, the molecules being analyzed, and the solvents used.

Analytes with less interaction with the stationary phase or more interaction with the mobile phase will exit the column faster.

Main components of an HPLC system include the solvent reservoir, high-pressure pump, column, injector system, and detector.

The mobile phase is usually held in a reservoir and can be bubbled with helium to degas and avoid unstable baselines caused by dissolved air.

HPLC systems are mostly automated and controlled by computer, though manual injection of samples is still possible.

The injector or auto sampler introduces the sample into a phase stream that carries it into the high-pressure column for separation.

A detector captures the separated compound bands as they elute from the high-pressure column, sending data to a computer to generate a chromatogram.

Ultra High Performance Liquid Chromatography (uHPLC) uses columns with particles as small as 1.7µm and pressures exceeding 1000 bar, offering faster and more sensitive analyses.

uHPLC systems use smaller volumes of organic solvents, allowing more samples to be run in less time compared to standard HPLC.

Running uHPLC systems at pressures greater than 800 bar may cause quicker degradation of the columns.

Fast Protein Liquid Chromatography (FPLC) is similar to HPLC but specifically used for separating or purifying proteins and biomolecules.

FPLC columns operate at lower pressures (3-5 MPa), but most FPLC columns can be used in an HPLC system if pressure is limited.