Chapter 6 Flashcards
To memorise and score the test
What is the difference between liquid chromatography (LC) and high performance liquid chromatography (HPLC)?
In principle, LC and HPLC work the same way except that HPLC offers superior speed, efficiency, sensitivity, and ease of operation compared to traditional liquid column chromatography.
What are the fundamental concepts of HPLC?
- Understanding the different types/modes of HPLC
- Distinguishing it from other forms of liquid chromatography
- Identifying different chromatograms
- Troubleshooting issues that may arise during analysis.
How does HPLC separate compounds dissolved in a solution?
HPLC separates compounds by injecting a small volume of liquid sample into a column packed with tiny particles called the stationary phase. The components of the sample are moved down the column with a liquid mobile phase forced through the column by high pressure.
What are some common chromatography techniques used in HPLC and GC?
In modern analytical chemistry, HPLC and GC are the major techniques used. HPLC is suitable for nonvolatile species and offers high performance and speed compared to traditional liquid column chromatography. GC, on the other hand, uses a gas mobile phase and is suitable for volatile compounds.
What is the role of the mobile phase in HPLC?
As a carrier to the sample solution and is continuously applied to the column or stationary phase. It facilitates the separation of biomolecules by using two or more solvents in certain ratios
Why is it important to degas solvents/mobile phase in HPLC?
To remove impurities. Impurities may interfere with the detection system, especially absorbance below 200 nm. Degas methods include warming, vigorous stirring, vacuuming, ultrasonification, and bubbling helium gas through the eluent reservoir.
What is the difference between isocratic elution and gradient elution in HPLC?
Isocratic elution is a separation method where the mobile phase composition remains constant throughout the procedure. In contrast, gradient elution involves gradually changing the mobile phase composition during the sample run. Gradient elution decreases the retention of later-eluting components, resulting in narrower peaks and improved peak shape and height
What is the difference between normal phase and reverse phase chromatography in HPLC?
In normal phase chromatography, the stationary phase is polar (e.g., alumina or silica gel), while the mobile phase is non-polar (e.g., hexane). In reverse phase chromatography, the stationary phase is non-polar (e.g., C18) and the mobile phase is polar (e.g., water, methanol, acetonitrile). Non-polar compounds elute faster in normal phase, while polar compounds elute later. Reverse phase chromatography is more commonly used as drugs are usually polar (hydrophilic).
Why are rigid solid particles used as the stationary phase in HPLC?
Rigid solid particles, such as microporous, pellicular, or bonded phase particles, are used as the stationary phase in HPLC to reduce space for diffusion. These spherical particles with uniform size provide efficient separation
How does the mobile phase facilitate the migration of components in an HPLC assay?
As a sample solution flows through the column with the mobile phase, the components of that solution migrate according to the non-covalent interaction of the compound with the column. The mobile phase carries the sample solution through the column, allowing for separation based on these interactions
What are some degassing methods used for solvents in HPLC?
- Warming
- Vigorous stirring with a magnetic stirrer
- Vacuuming
- Ultrasonification
- Bubbling helium gas through the eluent reservoir
Why is gradient elution preferred for the analysis of complex samples?
The mobile phase composition gradually changes during the sample run. It decreases the retention of later-eluting components, resulting in narrower peaks and improved peak shape and height. This technique is often used in method development for unknown mixtures
What is the purpose of a guard column in HPLC?
To protect the main column from impurities
Why are smaller packing particles preferred in HPLC columns?
Smaller packing particles lead to better resolution, but they also require high liquid pressure because they resist flow
What is the role of the stationary phase in HPLC?
- Separates the sample components using various physical and chemical parameters
- Usually made of stainless steel and withstands high pressure caused by the pump