HPLC chromatography
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
🔬 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.
🌡️ 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.
🧪 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)
💡Mobile Phase
💡Stationary Phase
💡Analyte
💡Sample Retention Time
💡Polarity
💡Injector System
💡Detector
💡Chromatogram
💡Degassing
💡Ultra High Performance Liquid Chromatography (uHPLC)
💡Fast Protein Liquid Chromatography (FPLC)
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.
Transcripts
High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps
a sample mixture or analyte in a solvent (known as the
mobile phase) at high pressure through a column with chromatographic packing material (stationary
phase).
The sample is carried by a moving carrier gas stream of helium or nitrogen.
HPLC has the ability to separate, and identify compounds that are present in any sample that
can be dissolved in a liquid in trace concentrations as low as parts per trillion.
Because of this versatility, HPLC is used in a variety of industrial and scientific
applications, such as pharmaceutical, environmental, forensics, and chemicals.
Sample retention time will vary depending on the interaction between the stationary
phase, the molecules being analyzed, and the solvent, or solvents used.
As the sample passes through the column it interacts between the two phases at different
rate, primarily due to different polarities in the analytes.
Analytes that have the least amount of interaction with the stationary phase or the most amount
of interaction with the mobile phase will exit the column faster.
Main components in an HPLC system include the solvent reservoir, or multiple reservoirs,
a high-pressure pump, a column, injector system and the detector.
The reservoir holds the solvent, which is referred to as the mobile phase because it
moves.
There are usually a minimum of two reservoirs in a system, with each holding up to 1000
cc of solvent and usually fitted with a gas diffuser through which helium can be bubbled.
A pump is used to generate a specified flow of the mobile phase.
Although manual injection of samples is still possible, most HPLCs are now fully automated
and controlled by computer.
The injector, or auto sampler, introduces the solvent into a phase stream that carries
the sample into the high pressure (up to 400 bar) column, which contains specific packing
material needed to effect separation.
The packing material is referred to as the stationary phase because it is held in place
by the column hardware.
A detector is needed to see the separated compound bands as they elute from the high
pressure column.
The information is sent from the detector to a computer which generates the chromatogram.
The mobile phase exits the detector and is either sent to a waste, or collected, as desired.
Helium sparging is an effective method of degassing the mobile phase to avoid unstable
baselines caused by dissolved air.
Nitrogen is used as a nebulisation gas in Evaporative Light Scattering Detector (ELSD)
where the solvent
is evaporated from the sample leaving a mist as is measured.
High-Performance Liquid Chromatography - Other HPLC Types
Ultra High Performance Liquid Chromatography (uHPLC):
Where standard HPLC typically uses column particles with sizes from 3 to 5�m and pressures
of around 400 bar, uHPLC use specially designed columns with particles down to 1.7�m in
size, at pressures in excess of 1000 bar.
The main advantage of an uHPLC is speed.
These systems are faster, more sensitive, and rely on smaller volumes of
organic solvents than standard HPLC, resulting in the ability to run more samples in less
time.
However, if the systems are run at typical pressures greater than 800 bar, the columns
age, or degrade quicker.
Newer technology is being developed for uHPLC units to use column particles with 1�m size,
and pressure potentials up to 6,800 bar.
uHPLC is also known as UPLCTM, a trademark of the Waters Corporation.
Fast protein liquid chromatography (FPLC): FPLC is
a system similar to high-performance liquid chromatography that
is
used to separate or purify proteins and other biomolecules from complex mixtures.
The main difference between FPLC and HPLC is
the standard working pressure.
FPLC columns can only be used
up to maximal pressures of 3-5 MPa.
If the pressure of the HPLC can be limited, nearly
every FPLC column
may also be used
in an HPLC system.
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