Chromatography 1

Question 1

Chromatography

Answer 1

This is the science of separating mixtures. The process works because different molecules have different properties (size, mass, polarity etc.)

The molecules travel at different rates, this is utilised to separate the components of the mixture.

Question 2

Early Chromatography (1)

Answer 2

A Russian biologist first developed modern chromatography from paper chromatography.

In column chromatography, a solution is poured into a tube packed with stationary phase with different parts of the solution exiting at different times.

Question 3

Mobile and Stationary Phases

Answer 3

Separation occurs as the sample in question is transported by a mobile phase, with this passing over or through a stationary phase.

Some components are strongly held by the stationary phase and travel slowly.

Other components are weakly held by the stationary phase, and travel more quickly.

Question 4

Separation

Answer 4

Separation occurs based on the differential affinity to the stationary phase.

At first, both components are together. Gradually the different rates of the travel spread the mixture.

Until the two components travel as separate components. It is fully separated components which are then used.

Question 5

Absorption Chromatography

Answer 5

There is competition between the components of the mixture for sites to absorb in the stationary phase.

The main property which effects separation is the differences in polarity of the components in the mixture.

The stationary phase can be polar or non-polar, and is normally a solid. The shapes of molecules can also have an influence.

Question 6

Partition Chromatography

Answer 6

Differences occur with partition chromatography based on the mobile phase used.

The mobile phases can be either a gas or liquid. The components of the mixture travel when dissolved in the mobile phase and are temporarily immobile when attached to the stationary phase.

Basically the more time spent with the mobile phase the faster the rate of travel for that component.

Question 7

Gas Chromatography

Answer 7

Lower boiling point components spend more time present as a gas and move rapidly with the mobile phase.

Higher boiling point components will spend more time as a liquid retained by the stationary phase.

Question 8

Gas Chromatography - Gas/Liquid

Answer 8

Mobile phase - inert gas (hydrogen, helium or nitrogen)

Stationary phase - high molecular liquid (bonded to a support of the wall of the column)

Question 9

Gas Chromatography - Gas/Solid

Answer 9

Mobile phase - inert gas (hydrogen, helium or nitrogen)

Stationary phase - solid (often porous and absorbent, typically used for components of air etc.)

Question 10

Liquid Chromatography

Answer 10

Those more soluble in the mobile phase, will spend more time dissolved in the mobile phase and travel faster.

Conversely those more soluble in the stationary phase will travel slower.

Question 11

Liquid Chromatography - Normal Phase

Answer 11

Mobile phase - non-polar organic solvents (costly to purchase and dispose of)

Stationary phase - high polar

Typically results in the least polar components of the analyte mixture having the highest rate of travel and eluting first.

Question 12

Liquid Chromatography - Reverse Phase

Answer 12

Mobile phase - highly polar ofteb aqueous based (relatively cheap in all respects)

Stationary phase - non-polar

Typically results in the most polar components of the analyte mixture having the highest rate of travel and eluting first.

Question 13

Size Exclusion Chromatography

Answer 13

Separation is based on the physical sizes of molecules. The stationary phase usually consists of a porous material containing many small holes.

When a molecule is larger than the size of the pore it passes around the porous material and travels quickly.

Smaller molecules enter the pores, this causes the molecule to take longer to travel through the stationary phase.

Question 14

Uses of Chromatography - Food Testing

Answer 14

HPLC-MS was successfully used to find out if meat labelled as beef was beef, horse or a mixture.

Question 15

Uses of Chromatography - Beverage Testing

Answer 15

GC is used to determine ethanol content by makers of alcoholic beverages, other companies use it to monitor the sugar content.

Question 16

Uses of Chromatography - Fuel

Answer 16

GC is ideal for analysing volatile components of petrol and diesel, enabling them to produce the different mixtures needed in winter and summer.

Question 17

Chromatographic Equipment

Answer 17

Chromatography types are separated by the equipment/instruments being used.

Question 18

Thin Layer Chromatography - TLC

Answer 18

Qualitative only.

Applications:
Drugs and poisons
Inks, dyes and cosmetics
Oils, waxes and greases

Question 19

Gas Chromatography - GC

Answer 19

Quantitative and qualitative.

Applications:
Drugs and poisons
Explosives
Fire accelerants
Oils, waxes and greases

Question 20

High Performance Liquid Chromatography - HPLC

Answer 20

Quantitative and qualitative.

Applications:
Drugs and poisons
Inks, dyes and cosmetic
Oils and greases

Question 21

TLC (1)

Answer 21

The stationary phase is typically cellulose or silica gel adhered to a plastic sheet.

Allows the use of a wider range of mobile phases.

Identification is done by comparing the travel of components of the mixture analysed.

Standards are often separated under the same conditions to ensure identification is correct.

Question 22

TLC (2)

Answer 22

TLC can be used as a confirmatory technique, but is normally restricted to being used to assist the possible identification of presumptive tests, prior to confirmatory tests.

Question 23

TLC - Inks, dyes and Cosmetics

Answer 23

The spots generated by the separated components may be highly visible, allowing the distance travelled to easily be measured.

Question 24

TLC - Drugs, Greases etc.

Answer 24

Methods are needed to visualise the different spots, which may require a spray on stain that chemically reacts to produce a visible spot.

Alternatively, certain chemicals fluoresce under UV light, allowing the position of spots to be recorded

Question 25

HPLC

Answer 25

The separation occurs in a pipe (column). This column is tightly packed with the stationary phase.

A high pressure pump is needed to ensure the mobile phase travels through the column. The pump carefully controls the flow of the liquid to ensure that the flow rate will be the exact same on each analysis run.

The sample is usually injected into the instrument via syringe and the flow pushes it into the column. Eventually the separated components are detected as they leave the column and enter a detector.

Question 26

GC

Answer 26

Separation can occur in either a column, tightly packed with the stationary phase, or in a fine capillary coated with the stationary phase.

The mixture of the mobile phase and sample enter the column where separation occurs on leaving the column.

This process occurs in an oven which allows the separation of the components of the mixture to be partly be controlled by changing the temperature.

Question 27

GC Benefits

Answer 27

Uses inert carrier gas (mobile phase) that acts as a physical force pushing the components through the column.

Separation is normally optimised by controlling the temp of the column oven.

High temperature of the injector means it is unsuitable for thermally unstable material.

High resolution and capable of separating extremely complex mixtures of similar compounds.

Question 28

HPLC Benefits

Answer 28

Uses either a polar or non-polar solvent which interacts with the components of a mixture.

Separation may be controlled by alteration of the concentration, and the pH of the mobile phase.

Suitable for thermally unstable compounds, such as some drugs and explosives.

Lower resolution so only able to separate simple mixtures.

Question 29

Limitations

Answer 29

Samples typically have to be fluid, solid samples require dissolution before analysis.

Complex mixtures may require a long run time to allow full separation.

Question 30

Retention and Retardation Factor

Answer 30

Both expressions of how much a component is retained by the stationary phase.

Often this is seen as the time it takes for a component to travel through the stationary phase.

Allows comparison to literature data, making it possible to make tentative identifications without the need to analyse all compounds.

Question 31

Retardation Factor

Answer 31

In planar chromatography, the distance travelled by the component and the mobile phase are used.

Rf = rate of movement of the component/rate of movement of mobile phase

Question 32

Retention Factor

Answer 32

In column chromatography, the retention times for the component and the mobile phase are used.