Topic 4: Chromatography Flashcards
What is chromatography? How does it work?
- The separation of analytes based on differences in partition between a mobile phase and an immiscible stationary phase
- The sample mixture is applied to the column (or stationary phase) and then “washed” through the column by the mobile eluent. Rate at which the solute is washed from the column is dependent on the average time it spends partitioned into the mobile phase or visa versa. Solutes that bind to the stationary phase more strongly are retained longer.
Label the type of chromatography.
What does tR and tm mean?
TR means retention time, which is the time it takes an analyte to reach the detector after it is injected.
Tm is the dead time, which is the time for an unretained peak takes to reach the detector
What is the retention factor?
The migration rate of solutes on a column
What is selectivity factor (α)?
An indication of the relative selectivity of a column for different analytes. A and B selected such that alpha is always greater than unity.
What is plate height?
An indication of the efficiency of column in terms of length of column required for a degree of separation. H is inter-related to the length (L) of the column. Therefore, the number of theoretical plates per column (N) is also important. In order to measure , the Gaussian nature of a peak is used, from this H can be related to the variance
Explain the van Deemter equation. .
A= in a packed column the solute can travel by many routes, some of which are longer than other. Also called eddy diffusion
B/u= the longitudinal diffusion term=diffusion that occurs due to the concentration gradient int eh mobile phase
C=mass-transfer coefficient= the equilibrium action for solutes to go in between mobile and stationary phase.
What is the importance of solvent degassing in HPLC?
One critical parameter is the quality of the solvent used. Solvents must be free of particulate, contaminants and dissolved gases. Dissolved gases come out of solution and form micro-bubbles which can interfere with accurate flow and become lodged in detector cells, creating baseline instability.
Describe the three ways of solvent degassing.
External vacuum degassing: a simple and effective form of degassing is to hold a flask of mobile phase under a vacuum while agitating the contents using a stirrer or an ultrasonic bath.
Helium sparging: helium sparging is a simple technique. A helium tank is connected to the solvent reservoirs of the chromatograph, and helium is bubbled into the eluent. Because helium has very low solubility in most solvents, it displaces the more common gases from the mobile phase. This approach has two limitations; 1) large quantity of expensive helium gas required, and the potential exists for changing the composition of the premixed solvents by selectively volatilizing the more volatile solvent
On-line Degassing: a vacuum is drawn on semipermeable tubes through which the eluents run. The vacuum draws air from the flowing solvents and discards it to waste. .
What is isocratic?
Eluent has a constant composition throughout the analysis. This simplifies system requirements and set-up, however, good resolution occurs only for a limited set of analytes
What is gradient system?
Eluent composition changes during the collection of a chromatograph. A series of solvent systems can be mixed together and this mix varied in a gradual manner, effectively altering the solvation ability of the mobile phase. Generally, two-binary, three-ternary or four-quaternary, solvents systems can be mixed. However, binary systems are the most common.
What is the normal phase and reverse phase packing?
- Normal phase
- Mobile phase is non-polar or less polar solvent
- Stationary phase is polar
- Reverse phased
- Stationary phase is non-polar or weakly polar
- Mobile phase is polar
