21 Questions
What is the function of the solvent reservoir in HPLC?
To provide a flow of the mobile phase through the HPLC injector, column, and detector
What are the requirements of a standard HPLC pump?
Flow rate ranging from 0.1 to 10 ml/min and constant pressure
What is the main difference between isocratic and gradient elution?
The mobile phase composition remains constant throughout the separation process in isocratic elution, while it changes in gradient elution
Which type of elution leads to shortening the analysis time and reducing tailing for sharp peaks?
Gradient elution
What is a common function of the filtration system in the solvent reservoir?
To prevent damage to the pump, injector valve, or clogging of the column
What does the pump in HPLC provide?
A flow of the mobile phase through the HPLC injector, column, and detector
What is the purpose of decreasing the retention of the last eluting peak in HPLC?
To decrease the elution time of the last peak
What is the function of a fixed-volume loop in HPLC?
To introduce samples with high precision into the HPLC system
What material are HPLC columns typically made from?
Stainless steel
What is the purpose of a guard column in HPLC?
To protect the analytical column from damage
What type of surface does a reversed-phase stationary phase have in HPLC?
Relatively nonpolar surface
What is the ideal characteristic for the response time of a column in HPLC?
Independent on flow rate
What characteristic does an ideal HPLC column have in terms of stability and reproducibility?
Good stability and reproducibility
What is the purpose of frits at the ends of an HPLC column?
To keep the particles in the column
What is a key requirement for an HPLC column in terms of sensitivity?
Adequate sensitivity for the particular task
What is the principle behind UV Absorption Detectors in HPLC?
Absorption of ultraviolet or visible light
According to Beer’s Law, what does 'A' represent in the equation A = -log(I/Io) = εbC?
Absorbance
For which type of compounds are UV Absorption Detectors in HPLC best suited?
Compounds with conjugated double bonds or aromatic groups
What type of solvents are required for UV Absorption Detectors in HPLC?
Solvents that absorb poorly in UV
What is the main advantage of fixed wavelength UV Absorption Detectors in HPLC?
Absorption at a single wavelength
What type of compounds have a poor response with UV Absorption Detectors in HPLC?
Compounds with few or weakly absorbing functional groups
Study Notes
- The text discusses the key components and processes in High Performance Liquid Chromatography (HPLC).
- The HPLC system consists of a solvent reservoir, a pump, a sample injection system, columns and stationary phase, and detectors.
- The solvent reservoir holds the mobile phase, typically contains a filtration system, and may have a degasser.
- The pump provides a consistent flow of the mobile phase, generating pressures up to 6000 Ibs/in2 and maintaining pulse-free output.
- HPLC is mainly used with packed columns, which are made from stainless steel and have lengths between 3-50 cm and diameters between 3-10 mm.
- Columns are filled with stationary phases, like silica gel, and have porous materials to allow sample interaction and separation.
- The sample injection system is responsible for introducing samples into the HPLC system, using a fixed-volume loop (1 - 200 µl).
- HPLC columns have different surface polarities, like silica gel or derivatized silica gel, and can be used with polar or non-polar stationary phases.
- The ideal characteristics of HPLC columns include high sensitivity, good stability, wide linear dynamic range, short response time, insensitivity to changes, and ease of use.
- UV Absorption Detectors are the most common type in HPLC systems, using the absorption of ultraviolet or visible light according to Beer's Law.
- UV Absorption Detectors require solvents that absorb poorly in UV, and have different sensitivities and selectivities depending on wavelength.
- The detectors can be fixed wavelength or variable wavelength, using monochromators to select a specific wavelength.
Test your knowledge about the function and components of a solvent reservoir in liquid chromatography systems. Learn about filtration systems, degassers, and the role of the pump in providing a flow of the mobile phase.
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