1. BASED ON POLARITY

2. Paper Chromatography

3. What is Chromatography?
Chromatography is a technique for separating mixtures into their components
Identify
Separate
Mixture
Components

4. Uses for Chromatography
Real-life examples of uses for chromatography:
Hospital – detect alcohol/drug levels in blood
Police – to compare a sample at a crime scene to a suspect sample
Environmental Agency – find pollutants in the water supply

5. Definition of Chromatography
Detailed Definition:
Chromatography separates components (parts) within a mixture by
using the differential affinities of the components for the mobile
medium and for the stationary medium (absorbing)
Terminology:
Differential – difference Affinity – attraction (polar or nonpolar)
Mobile Medium – the moving part (solvent, like water or alcohol). The
liquid (or gas) that carries the mixture.
Stationary Medium – the part that does not move (the paper or gel)
What is important is how much they adsorb on the stationary phase versus
how much they dissolve in the mobile phase

6. 2 TYPES BASED ON MOBILE PHASE
CHROMATOGRAPHY
2 TYPES BASED ON MOBILE PHASE
LIQUID
PAPER
TLC
HPLC
GAS
CHR A D F

7. TLC AND PAPER

8. Principles of Paper Chromatography
Capillary Action – the movement of liquid within the spaces of a porous material. The paper’s plant cells themselves are made of cellulose and are which is polar have water in them and between them which is polar.
Due to electric forces he liquid is able to move up the filter paper because the attractions is stronger than the force of
gravity. Separation of components depends on both their solubility in the mobile phase and their affinity to the stationary phase.

9. Thin Layer Chromatography (TLC)

10. Illustration of Chromatography
Stationary Phase
Separation
Mobile Phase
Mixture
Components
Components
Affinity to Stationary Phase
Affinity to Mobile Phase
Blue
Cannot say (no movement)
Insoluble in Mobile Phase
Black
✓✓✓✓✓✓ ✓
Red
✓✓✓
Yellow ✓😀

11. Visualizing Agents
The sample (analyte) when it separates may not be visible, so a dye is used (already impregnated on the TLC gel) or UV light. Ex ninhydrin
purple

12. Rf = distance moved by substance distance solvent front
Substances that are soluble in the liquid
the Rf will be close to , as it heads to the top
For substances that are rather insoluble in the liquid Rf
will be close to , they don’t move
Thus all numbers must never be more than 1 for Rf
Notice Retardation factor (above) is based on distance for
Paper and TLC.
Next we visit HPLC and GLC which will not measure
Distance, rater the measure retention time spent inside a column.

13. Column Chromatography
Adsorbents:
Alumina & Silica gel (is porous sand) in which more polar molecules are adsorbed more strongly & thus, will elute more slowly

14. COLUMN CHROMATOGRAPHY
Stationary phase silica
Mobile phase suitable organic solvent
Separation components interact with the stationary
phase to different extents
Method
• a chromatography column is filled with solvent and silica
• drops of the mixture (analyte) are placed on top of the silica - A
• the tap is opened to allow the solvent to flow out
• components travel through at different rates and separate - B
• batches of solvent are collected at intervals - C
• the solvent in each batch is evaporated to obtain components A B B C

15. HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC)
A better form of column chromatography. Instead of draining down through the stationary phase, the solvent is forced through under high pressure.
Stationary phase silica (tiny solid micro beads)
Mobile phase suitable solvent
Separation similar to column chromatography
Alumina is also used (Al2O3) with Iron impurities you get a sapphire
And chromium impurities = ruby

16. B A C D HPLC Column Detector Mixture of compounds Column Injector
Made by RW

17. Detector
Injector
Column D A C B

18. Detector D A C B
Injector
Column
Different substances stick to the column and pass through at different speeds.
Amount
The detector makes a graph
Time

20. Each peak is a chemical in the mixture. C eluted first.B B C A

21. HLPC

22. GAS LIQUID CHROMATOGRAPHY (GLC)
Stationary phase liquid adsorbed on an inert solid support
Mobile phase gas
Method
a sample is injected and vaporized
then carried along by an inert gas
column contains a liquid stationary
phase, adsorbed onto an inert solid
the time taken to travel through the tube will depend on how much time is
spent moving with the gas rather than being attached to the liquid.

23. GAS LIQUID CHROMATOGRAPHY (GLC)
Detection one method is an FID - flame ionisation detector
The FID • as a component exits, it is burned in a hydrogen flame
• ions are produced
• detected as the ions produce an electric current
• the greater the amount = larger current
• the current can be represented by a chromatogram

24. GLC

25. What happens in practice.
Compounds that have high affinity for mobile phase emerge first, (most volatile).
Chromatogram charts recorder response against time.
Retention time – characteristic of the compound under given conditions.

26. GAS LIQUID CHROMATOGRAPHY (GLC)
Interpretation
• the areas under the peaks are proportional to the amount of a compound
• retention times are used to identify compounds – they are found out by
putting known compounds through the system under similar conditions
and comparing the knowns to the sample. If they match we have identified
the chemical
The area under a peak is proportional to the amount present.
Each component has a different retention time.

27. Summary 4 Types of Chromatography
Stationary Phase
Moblie Phase
Sample
Paper (Liquid chromo)
Paper (solid)
Cellulose, coupled with water absorbed within the fibers)
Polar and nonpolar mixture of liquid solvents
Dried onto stationary paper
TLC (Liquid chromo)
Silica (solid, SiO2)
Dried onto stationary gel
HPLC (Liquid chromo)
Silica (solid) and solvent slurry
Dissolved into the mobile phase of the gas
Gas (Gas chromo)
Beads (solid and Liquid waxy polymer(
Inert gas (N2, Hel
Heated to a Gas to go with N2 or He

28. Interesting $1 Centrifuge