Chromatography

Question 1

What is chromatography?

Answer 1

A method of separating soluble substances by their partition between two phases

Question 2

What two things are needed for all forms of chromatography?

Answer 2

Stationary and mobile phase

Question 3

Name the three main types of chromatography

Answer 3

Thin layer chromatography
Column
Gas

Question 4

What are the mobile and stationary phases in TLC?

Answer 4

Mobile - solvent

Stationary - SiO2 or Al2O3 on a glass or plastic plate

Question 5

Is the TLC plate polar or non-polar?

Answer 5

Polar

Question 6

Outline the method of doing TLC

Answer 6

1. Prepare TLC plate and draw pencil line as baseline
2. Use capillary tube to dot samples onto the plate
3. Put plate into the solvent with the baseline above the solvent
4. Put on a lid and allow to run for half an hour
5. Draw line on solvent front and measure it’s distance from baseline
6. Identify the spots and measure their distance from BL
7. Calculate Rf values and compare them to standard runs

Question 7

Why do you hold the TLC plate with gloves?

Answer 7

So it doesnt get contaminated

Question 8

Why do you use a pencil to draw the lines in TLC?

Answer 8

Bc it’s insoluble and pen contains substances which would run up the plate and would affect the results

Question 9

Why don’t you submerge the line in the solvent in TLC?

Answer 9

So that the samples and the line don’t dissolve

Question 10

If the spots in TLC are colourless, how do you identify them?

Answer 10

UV light
Ninhydrin - developing agent
Iodine - stains it brown
Potassium manganate

Question 11

How does TLC separate compounds?

Answer 11

Adsorption

Question 12

How does column chromatography separate compounds?

Answer 12

Adsorption

Question 13

What are the stationary and mobile phases in column chromatography?

Answer 13

Stationary phase - silica or alumina

Mobile phase - non-polar solvent e.g. hexane

Question 14

Define retention time

Answer 14

The amount of time that a substance remains in the column

Question 15

What does assayed mean?

Answer 15

Analysis

Question 16

Summarise how column chromatography works

Answer 16

1. SP poured into column and air bubbles removed
2. Mobile phase then added and equilibrated
3. Sample dissolved in a small bit of solvent poured through
4. Sample follows mobile phase through column and substances begin to separate
5. Separated compounds colllected in fractions as they elute from the column

Question 17

If a substance is eluted later in column chromatography, what can be said about its polarity?

Answer 17

It’s polar because it has a greater affinity for the stationary phase so it adsorbs to the stationary phase more

Question 18

How does separation occur in gas-liquid chromatography?

Answer 18

Partition

Question 19

What are the stationary and mobile phases in GC?

Answer 19

Stationary - non-volatile liquid such as a waxy polymer

Mobile - Inert gas

Question 20

Name potential mobile phases for GC?

Answer 20

Argon, nitrogen and helium

Question 21

What size is the column in GC?

Answer 21

1-4m long and about 4mm wide

Question 22

Explain the process of gas chromatography

Answer 22

1. Sample injected using a needle into carrier gas and heated
2. Gaseous sample swept through column
3. Column is in an oven and heated to temp lower than the gas
4. Some gases condense into stationary phase, others are carried through in the gas phase
5. When eluted, it can be detected by mass spec or by flame ionisation detector

Question 23

In GC, are the less volatile substances retained for a longer or shorter time and why?

Answer 23

Longer bc they can condense onto or dissolve into the stationary phase

Question 24

In GC, are the more volatile substances retained for a longer or shorter time and why?

Answer 24

Shorter because they stay in the gas phase and pass straight through

Question 25

In GC, what is the temperature of the column oven?

Answer 25

50-250 degrees so that it's slightly lower than the injector oven so some gases can condense and be retained

Question 26

How is the information from the gas chromatogram analysed?

Answer 26

A graph of retention time against relative intensity is plotted. Relatve amount of each substance can be found and compared with standards under the same conditions Put through a mass spec

Question 27

What does the area under the peak in GC tell?

Answer 27

The relative amount of each substance

Question 28

What three things may happen to a molecule in GC?

Answer 28

Condenses onto the stationary phase Dissolves into the stationary phase Stays in the gas phase

Question 29

How can GC and MS be used to analyse a mixture of tripeptides?

Answer 29

GC separates the substances Mass spec has a distinctive fragmentation pattern Compare m/z values to database

Question 30

Explain how you could use a gas-liquid chromatogram to determine the percentage composition of each component in a mixture

Answer 30

Chromatogram has retention time against relative intensity Find area under each peak Find total area Do area under peak/total area x100

Question 31

What is partition chromatography?

Answer 31

Separation when the stationary phase is liquid (substance can partition between the phases). Distribution between SP and MP

Question 32

What is adsorption chromatography?

Answer 32

When the stationary phase is solid e.g. TLC

Question 33

What things happen to a molecule being separated in GC chromatography?

Answer 33

Stays as a gas Condenses to a liquid onto the stationary phase Dissolves in the liquid on the SP

Question 34

What factors does the retention time depend on?

Answer 34

temperature of the column boiling point solubility in liquid phase

Question 35

Why do different amino acids have different Rf values?

Answer 35

Different polarities | Different retention by SP or diff solubility in the MP