Chapter 10E - Chromatography

Question 1

What is Chromatography?

Answer 1

• seperate/identify/quantify components of a mixture
• seperates substances based on solubility
• polar molecules dissolve in polar solvents vice versa

Question 2

Stationary Phase

Answer 2

• solid
• fixed position (not moving)
• can be polar/non-polar

Question 3

Mobile Phase

Answer 3

• moving phase (fluid)
• can be polar/non-polar

Question 4

How does Chromatography work?

Answer 4

• mobile phase sweeps over stationary phase, carrying components of the mixture
• components seperate based on different relative attractions (adsorption) to the stationary phase and readiness to dissolve (desorption) in mobile phase

Question 5

Rate of movement depends on

Answer 5

• how strongly compound adsorbs onto the stationary phase
• how readily it dissolves (desorbs) onto the mobile phhase

Question 6

How does column chromatography work?

Answer 6

1. sample is placed at the top of column
2. mobile phase moves down column
3. components seperate based on adsorption/desorption
4. components elute from the column at different rates

Question 7

Retention time

Answer 7

Time taken for a component to pass through the column

Question 8

Larger retention time

Answer 8

• components more strongly adsorbed to the stationary phase
• has polarity closest to stationary phase

Question 9

Smaller retention time

Answer 9

• components least strongly adsorbed onto the stationary phase
• component has polarity closest to that of the mobile phase

Question 10

What is HPLC

Answer 10

• qualitative & quantitative technique
• mobile phase/sample pumped through tightly packed stationary phase under pressure

Question 11

Normal phase HPLC

Answer 11

• polar stationary phase

Question 12

Reverse phase HPLC

Answer 12

• non-polar stationary phase

Question 13

Detector

Answer 13

• beam of light is passed through the sample
• recorded light produces a chromatogram

Question 14

Factors affecting retention time

Answer 14

• identity of SP
• identity of MP
• length of column
• temperature of column
• mobile phase flow rate
• surface area of SP

Question 15

Qualitative Analysis

Answer 15

• retention time
• position of the peak
• identify components of a mixure (compare time w/ known standards run under same operating conditions)

Question 16

Quantitative Analysis

Answer 16

• area under the peak
• uses a calibration curve
• compare areas w/ areas for standard samples
• higher concentration = greater area (retention time remains the same)

Question 17

Calibration curves

Answer 17

1. several standard solutions of component of known concentration are run through the HPLC column under identical conditions
2. resulting peak areas are then plotted against concentrations to produce a calibration curve
3. the concentration of the component in the mixture is interpolated from the calibration curve

Question 18

What is the affect of pressure on retention time?

Answer 18

• mobile phase normally under high pressure (improved peak resolution)
• decrease: longer retention time
• particles take more time to move through the column due to more interactions b/w particles + stationary phase.

Question 19

What is the effect of temperature on retention time?

Answer 19

Decreased temperature:
* particles move slower
* longer retention time
* substances will take more time to move through the column (more interactions b/w particles & stationary phase)

Question 20

What is the effect of column density on retention time?

Answer 20

A less tightly packed column:
* easier for particles to move through
* shorter retention time
* particles will take less time to move through the column (less interactions)

Question 21

Describe the stationary phase of HPLC

Answer 21

• small particle size
• frequent adsorption/desorption/seperation
• larger surface area