1. Chromatography
Chapter 6

2. Chromatography
A technique used to separate the substances present in a mixture.
Chromatography can be used for both qualitative and quantitative analysis
It can also be used to determine the identity of a substance.
Chromatography applications include the identification of:
Drugs present in blood
Sugars in fruit juice
Hydrocarbons in oil
Pollutant gases in exhaust fumes

3. How does chromatography work?
There are many different methods of chromatography, but all methods have:
A stationary phase
A mobile phase (or moving phase)
Turn to page 62 and look at figure 6.1
What do you think is the stationary phase?

4. How does chromatography work?
Different components of a mixture will be attracted (adsorb to) the stationary and mobile phases to different extents.
Chromatography uses these differences in attraction to separate the components.
The moving fluid, referred to as the mobile phase, passes over the stationary phase.
The components of the mixture are separated according to their relative attractions to the mobile and stationary phases.

5. How does chromatography work?
The rate of movement of each component depends mainly upon
How strongly it adsorbs onto (is attracted to) the stationary phase.
How readily it dissolves in the mobile phase

6. Paper and Thin-layer chromatography (TLC)
TLC is similar to paper chromatography which you have done in junior science
Paper chromatography uses high quality absorbent paper (filter paper) as the stationary phase
TLC uses a thin layer of fine powder such as alumina, spread on a glass or plastic plate
Both paper and TLC are good for qualitative analysis only.

7. TLC - Procedure A solution of the sample to be analysed is made up
As small a spot as possible is place onto the end of the chromatography plate
This spot is called the origin
The plate is then placed into a container with the edge of the plate submerged in solvent (the origin should be above the solvent)
As the solvent rises up the plate the components of each sample separate

8. TLC
The different components of a mixture will move at different rates depending on relative strength of attractions to the stationary and mobile phases.
What is more strongly attracted to the stationary phase?

9. Interpreting chromatograms of TLC
A chromatogram is the pattern of bands or spots formed on the plate in TLC
The identity of the chemicals in the mixture can be identified in two ways
1. running standards of known chemicals on the same chromatogram as the unknown sample
2. calculating Rf values of the samples
Which method would be more effective??

10. Rf values Distance moved from origin by component Rf =
Distance moved from origin by solvent
Rf values will always be less than one
The component most strongly adsorbed onto the stationary phase moves the shortest distance and has the lowest Rf value
By comparing the Rf values of components of a mixture with the Rf values of known substances under identical conditions, the compounds present in a mixture can be identified
What issues can occur with this method

11. Your Turn
Page 64
Question 1
Page 72
Question 5b

12. Column Chromatography
The stationary phase is a solid, or a solid that has been thinly coated in a viscous liquid and packed into a glass column.
The sample is applied carefully to the top of the packing and a solvent, which acts as the mobile phase, is dripped slowly on to the column from a reservoir above.
A tap at the bottom of the column allows the solvent, which is called the eluent, to leave the column at the same rate as it enters it at the other end.
There are two techniques based on column chromatography, high performance liquid chromatography (HPLC) and gas chromatography.

13. HPLC and GC
HPLC and GC chromatograms represent the qualitative and quantitative aspects of chromatography.
Number of peaks – mixture separated into three different compounds
Retention time – identifies each component – qualitative analysis
Area under each peak – relative amount of each compound – quantitative analysis
Retention time (Rt)

14. High performance liquid chromatography (HPLC)
This method is used routinely for pharmaceutical and industrial analysis.
It allows extremely sensitive analysis of a wide range of compounds.
It can be used to detect barbiturates in the blood.

15. HPLC
There are many ways in which HPLC differs from tradition chromatography:
The size of the particles in the solid stationary phase is often times smaller
The very small size of these particles allows for more frequent adsorption and desorption of the components, giving better separation
The small particles size creates considerable resistance to the flow of the mobile phase so the solvent is pumped through under high pressure
A range of solids are available for use in HPLC columns, some with chemicals specially bonded to their surfaces to improve the separation of particular classes of compounds

16. HPLC The detector is usually a UV light
Many organic compounds absorb UV light
The amount of light received by the detector is recorded on a chart that moves at constant speed
This resulting trace is the chromatogram
The time taken for a component to pass through the column is called the retention time, Rt
The Rt are used to identify the components associated with the peaks on a chromatogram.
The relative amounts of each component in a mixture may be determined by comparing the areas under each peak with areas under peaks for standard samples

17. Gas Chromatography (GC)
This is the most sensitive chromatographic technique
It is capable of detecting as little as 10-12g of a compound
It is limited to compounds that can be easily vaporised without decomposing.
Urine samples taken from athletes for drug tests are analysised by GC

19. GC Gas chromatography has the following features
The mobile phase is a gas, usually nitrogen, called the carrier gas
A small amount of sample is injected into the top of the column through an injection port
The injection port is heated to a temperature sufficient to instantly vaporise the sample, which is then swept into the column by the carrier gas.
The column is in a loop, this is because of the fast moving gaseous phase. So the column must be longer than in HPLC to allow for effective interaction with the stationary phase
The column is mounted in an oven and heated

20. Interpreting Chromatograms
The time a component takes to pass through the column is called the retention time, Rt.
The same compound will give the same retention time if the conditions (temp, mobile phase, stationary phase, flow rate, pressure etc) remain the same.
Each component forms one peak, however it is possible for a number of peaks to coincide and be indistinguishable.
Look at page 69.

21. Figure 6.11 Gas chromatogram of a petrol sample.
Figure 6.12 Chromatogram of a reference sample containing a mixture of butane, 2-methylbutane, hexane, benzene and 2-methylhexane.
Figure 6.13 Petrol sample spiked with benzene.

22. Worked Example 6.3
Page 69
What is the concentration of the sample of benzene?
Your Turn: Try question 14 on page 74

23. Chapter homework Page 72 Question 7 and 9 Page 74 Question 17 Page 75