Chromatography

Question 1

What does the mobile phase do?

Answer 1

The mobile phase moves through the system and carries sample components

Question 2

What does the stationary phase do?

Answer 2

The stationary phase is held within the system and does not move

Question 3

What will be the effect on a compound of a stronger interaction with the stationary phase?

Answer 3

This will cause the compound to be retained by the stationary phase and move more slowly throughout the column

Question 4

What is the plot created by a chromatograph called?

Answer 4

Chromatogram

Question 5

What is retention time (t(R))?

Answer 5

The average time or volume that is required for a particular chemical to pass through a column

Question 6

What is elution time (t(E))?

Answer 6

The time between the start of the seperation and when the elute gone through the system

Question 7

What is the area or height of the peak used for?

Answer 7

Measure the amount of the compound present

Question 8

What is the retention time of the peak used for?

Answer 8

Identifying the eluting compound

Question 9

How does adsorption chromatography work?

Answer 9

Solid stationary phase and liquid or gasous mobile phase. The analyte is absorbed on the surface of the stationary and moves more slowly.

Question 10

How does partition chromatography work?

Answer 10

A liquid stationary phase is bonded to a solid surface and analyte equilibrates between stationary and mobile phase.

Question 11

How does ion-exchange chromatography work?

Answer 11

Anions or cations are covalently attached to the stationary phase. Analytes of the opposite charged is attracted to the charged stationary phase.

Question 12

How does molecular (or size) exclusion chromatography work?

Answer 12

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

Question 13

What does a larger retention factor mean?

Answer 13

Larger k -> longer retention time -> More retained/ more time spent i stationary phase

Question 14

What does a larger separation factor indicate?

Answer 14

Larger seperationfactor => Greater separation between compounds

Question 15

What two factors affect resolution?

Answer 15

• Distance between peaks

- Width of the peaks

Question 16

What three phenomena cause band broadening?

Answer 16

• Multiple flow paths
• Longitudinal diffusion
• Resistance to mass transfer

Question 17

How does multiple paths effect band broadening and how can it be reduced?

Answer 17

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

Question 18

How does longitudinal diffusion work and how can it be reduced?

Answer 18

Compounds randomly diffuse along the length of the column in all directions (including up in the column). This gets reduced with increased flow rate.

Question 19

How does resistance to mass transfer work and how can the broadening be reduced?

Answer 19

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.

Question 20

What is a isotherm graph and what is it used for?

Answer 20

Graph with Cs plotted against Cm at a given temperature. Illustrates assymetric bandshapes

Question 21

What are assympetric bandshapes caused by and what effects are there?

Answer 21

Cause: Overloading
Result: Fronting or tailing

Question 22

What does fronting mean and which type of chromatography is this a problem?

Answer 22

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.

Question 23

How does tailing happen and how can you prevent it?

Answer 23

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.

Question 24

What three factors affect the resolution?

Answer 24

• Plate number (N)
• Separation factor (alfa)
• Retention factor (k)

Increase in either of these and the resolution will increase

Question 25

What effect does a low plate height have?

Answer 25

It creates narrow peaks and good resolution

Question 26

What effect does a low N value have on the peaks?

Answer 26

The peaks come out broad

Question 27

What kind of chromatography has the following characteristics?

Ions in mobile phase are attracted to counter-ions covalently attached to the stationary phase

Answer 27

Ion-exchange chromatography

Question 28

What kind of chromatography has the following characteristics?

Solute in mobile phase is attracted to specific groups covalently attached to stationary phase

Answer 28

Affinity chromatography

Question 29

What kind of chromatography has the following characteristics?

Solute equilibrates between mobile phase and surface of stationary phase

Answer 29

Adsorption chromatography

Question 30

What kind of chromatography has the following characteristics?

Solute equilibrates between mobile phase and film of liquid attached to stationary phase

Answer 30

Partition chromatography

Question 31

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.

Answer 31

Molecular exclusion chromatography