Chromatography Flashcards
What does the mobile phase do?
The mobile phase moves through the system and carries sample components
What does the stationary phase do?
The stationary phase is held within the system and does not move
What will be the effect on a compound of a stronger interaction with the stationary phase?
This will cause the compound to be retained by the stationary phase and move more slowly throughout the column
What is the plot created by a chromatograph called?
Chromatogram
What is retention time (t(R))?
The average time or volume that is required for a particular chemical to pass through a column
What is elution time (t(E))?
The time between the start of the seperation and when the elute gone through the system
What is the area or height of the peak used for?
Measure the amount of the compound present
What is the retention time of the peak used for?
Identifying the eluting compound
How does adsorption chromatography work?
Solid stationary phase and liquid or gasous mobile phase. The analyte is absorbed on the surface of the stationary and moves more slowly.
How does partition chromatography work?
A liquid stationary phase is bonded to a solid surface and analyte equilibrates between stationary and mobile phase.
How does ion-exchange chromatography work?
Anions or cations are covalently attached to the stationary phase. Analytes of the opposite charged is attracted to the charged stationary phase.
How does molecular (or size) exclusion chromatography work?
Analytes pass through a porous gel. The pores excludes larger molecules which therefor pass faster than small molecules. Importantly there should be no attraction between analyte and stationary phase
What does a larger retention factor mean?
Larger k -> longer retention time -> More retained/ more time spent i stationary phase
What does a larger separation factor indicate?
Larger seperationfactor => Greater separation between compounds
What two factors affect resolution?
- Distance between peaks
- Width of the peaks
What three phenomena cause band broadening?
- Multiple flow paths
- Longitudinal diffusion
- Resistance to mass transfer
How does multiple paths effect band broadening and how can it be reduced?
The peak gets broaden when the same compound can take multiple paths in a packed column. This gets reduced when you have smaller particles in the stationary phase
How does longitudinal diffusion work and how can it be reduced?
Compounds randomly diffuse along the length of the column in all directions (including up in the column). This gets reduced with increased flow rate.
How does resistance to mass transfer work and how can the broadening be reduced?
Mass transfer is when the analyte molecules get transfered between the stationary and mobile phase which is what creates separation between compounds. This transfer requires time which means that a slower flow rate will allow the compounds to be properly retained and broadening is reduced.
What is a isotherm graph and what is it used for?
Graph with Cs plotted against Cm at a given temperature. Illustrates assymetric bandshapes
What are assympetric bandshapes caused by and what effects are there?
Cause: Overloading
Result: Fronting or tailing
What does fronting mean and which type of chromatography is this a problem?
Fronting is caused when there is too much analyte in the stationary phase that the stationary phase begins to resemble the analyte. This makes the retention time increase which creates a gradual rise and an abrupt fall.
How does tailing happen and how can you prevent it?
Taling happens when you have polar analytes which interact with silanol groups in the stationary phase. A portion of the analyte molecules will interact more strongly with the stationary phase and are retained to a larger extent.
This can be reduced by silanization, by blocking the silanol groups with nonpolar trimethysilyl groups. This prevents the silanol groups to interact with the analyte.
What three factors affect the resolution?
- Plate number (N)
- Separation factor (alfa)
- Retention factor (k)
Increase in either of these and the resolution will increase
What effect does a low plate height have?
It creates narrow peaks and good resolution
What effect does a low N value have on the peaks?
The peaks come out broad
What kind of chromatography has the following characteristics?
Ions in mobile phase are attracted to counter-ions covalently attached to the stationary phase
Ion-exchange chromatography
What kind of chromatography has the following characteristics?
Solute in mobile phase is attracted to specific groups covalently attached to stationary phase
Affinity chromatography
What kind of chromatography has the following characteristics?
Solute equilibrates between mobile phase and surface of stationary phase
Adsorption chromatography
What kind of chromatography has the following characteristics?
Solute equilibrates between mobile phase and film of liquid attached to stationary phase
Partition chromatography
What kind of chromatography has the following characteristics?
Different-sized solute penetrates pores in the stationary phase to different extents. Largest solutes are eluated first.
Molecular exclusion chromatography