1. Chromatography

2. Learning Objectives Definition of Chromatography
Descriptions of Experimental Chromatography
Explaining Chromatographic separation
Types of Chromatography
Chromatographic separation
Choice of Solvent
Thin layer Chromatography
Column Chromatography
Paper Chromatography
Ion exchange Chromatography
Examples
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3. Learning Outcomes The student should be able to describe
How to use different chromatography method
Descriptions of Experimental Chromatography
Explaining Chromatographic separation
Types of Chromatography
Chromatographic separation
Choice of Solvents
Thin layer Chromatography
Column Chromatography
Paper Chromatography
Ion exchange Chromatography
Examples
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4. What is Chromatography?
A broad term that is used to describe a collection of laboratory techniques that are used to separate or analyze mixtures.
There are several different types of chromatographic methods including:
Gas chromatography
Paper chromatography
Thin-layer chromatography (TLC)
High pressure liquid chromatography (HPLC)
Affinity chromatography
Ion-exchange chromatography
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5. Chromatography Theory
Even though each method utilizes different techniques to separate compounds, the principles are the same.
Common to all:
Stationary phase
A solid or a liquid supported on a solid
Mobile phase
Liquid or gas
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6. Chromatography Theory
As the mobile phases passes through the stationary phase, it carries the components of the sample mixture with it.
The components of the sample will be attracted to the stationary phase, but there will also be a competing attraction for the mobile phase.
Each component will have its own characteristic balance of attraction to the mobile/stationary phase.
So they will not move at the same speed.
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7. Chromatography Theory WILL MOVE WITH THE MOBILE PHASE
The component that:
has a higher affinity for the stationary phase
lower affinity for the mobile phase
WILL LAG BEHIND
has a lower affinity for the stationary phase
higher affinity for the mobile phase
WILL MOVE WITH THE MOBILE PHASE
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8. Chromatography History
Paper chromatography: First analytical chromatographic technique developed. First published by Runge in 1855
1903: Russian botanist Mikhail Tswett
Separated plant pigments through column adsorption
chromatography
1922: L.S. Palmer used Tswett’s techniques on various natural products
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9. Thin Layer and Paper Chromatography History
But this method was really accepted until 1931, when Richard Kuhn used chromatography to separate isomers of polyene.
TLC History:
1938: Drop chromatography on horizontal thin layers was developed by Russian scientists N. A. Izmailov and M. S. Shraiber.
1950s: Justus G. Kirchner and his co-workers at the US Department of Agriculture's Fruit and Vegetable Laboratory in California perfected TLC.
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10. Thin Layer Chromatography

11. What is Mobile Phase?
The mobile phase is the part of the chromatographic system which carries the solutes through the stationary phase.
The mobile phases are either liquids or gases.
The liquid mobile phases are used to adjust the chromatographic separation and retention in liquid chromatography and the temperature of the gas mobile phase is used to adjust the retention in gas chromatography

12. Examples of Mobile Phase
Gas (GC)
Water (LC)
Organic solvent (LC)
Supercritical fluid (SCFC)
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13. What is Stationary Phase?
The stationary phase is the part of the chromatographic system though which the mobile phase flows where distribution of the solutes between the phases occurs.
The stationary phase may be a solid or a liquid that is immobilized or adsorbed on a solid. In general immobilization by reaction of a liquid with a solid is used in liquid chromatography and absorbtion of a liquid on a solid is used in gas chromatography but there are many exceptions to both of these generalizations.
The stationary phase may consist of particles (porous or solid), the walls of a tube (eg. capillary) or a fibrous material

14. Examples of Stationary Phase
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15. Thin Layer Chromatography
Stationary phase = A piece of glass, metal, or plastic coated with a thin, uniform layer of a solid adsorbent.
Usually silica gel (SiO2), alumina (Al2O3), or cellulose. A substance which fluoresces under UV light often incorporated into the stationary phase.
Zinc sulfide
Mobile phase= A suitable liquid solvent or mixture of solvents.
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16. TLC Procedure Step 1: Preparing the chamber
Choose a container that is large enough and can be sealed.
Add the a few cm of the mobile phase solvent to the chamber.
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17. TLC Procedure Step 1: Preparing the chamber
Seal the chamber and allow it to sit overnight if possible.
The atmosphere of the chamber should be saturated with the solvent vapors before running samples.
You may line part of the inside of your chamber with filter paper to aid in this saturation process.
Stops the solvent from evaporating as it rises up the stationary phase plate.
Allows for better development of the chromatograms.
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18. TLC Procedure Prepare the TLC plate:
Step 3: Preparing the stationary phase
Mix with:
Adsorbent (Silica gel powder)
Small amount of an inert binder (Zinc sulphide)
Water
Spread a thin layer (no more than a few mm) of the mixture on an non-reactive support.
After the plate is dried, it is activated by heating in an oven for approximately 30 minutes at 110˚C.
Prepare the TLC plate:
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19. TLC Procedure Step 4: Spotting the samples
TLC plates are also commercially prepared and can be purchased ready for use.
Draw a line of origin approximately 0.5 cm from the bottom of the filter paper.
Indicate where each sample will be added.
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20. TLC Procedure Step 4: Spotting the samples
If the sample isn’t in solution, dissolve it in an appropriate solvent.
As a rule of thumb, a concentration of about 1% (1g in 100ml) is good.
If the sample is too concentrated = smear
If the sample is too dilute = no results
Step 4: Spotting the samples
The image shows a sample ran at three different concentrations. The left plate was ran too concentration and the spots are running together. The other two plates yielded good separation.
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21. TLC Experimental Setup
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22. TLC Procedure Step 5: Developing the chromatograms
Allow the solvent to rise until it almost reaches the top of the plate.
Remove the plate from the chamber and mark the position of the solvent and front before it can evaporate.
If the sample spots are visible, mark their positions.
Step 5: Developing the chromatograms
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23. Visualizing Colorless Compounds
Step 7: Identify the spots and interpret the data.
What if the compounds being separated are colorless? How are the spots visualized?
Two ways to get around this problem:
Use fluorescence
Use chemical methods
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Final Chromatogram 23

24. Using Fluorescence to Visualize Spots
Substance which can fluoresce under UV light is added to stationary phase.
So, when the TLC plate is exposed to UV light, the entire plate will glow.
On the final chromatogram, the glow will be masked at positions were spots are located.
Examples: zinc sulfide
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25. Visualizing Spots Chemically
In some cases it may be possible to visualize the spots by reacting them with something that produces a colored product.
Iodine Crystals:
The dried chromatogram is placed into a closed container containing iodine crystals.
The iodine vapor either:
Reacts with the spots
Sticks more to the spots than it does to the rest of the chromatogram
The sample spots will be brownish in color.
The spots are more intense for unsaturated compounds
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26. Visualizing Spots Chemically
Ninhydrin:
The dried chromatogram is sprayed with a ninhydrin solution.
Reacts with amino acids to produce a colored product.
Mainly brown or purple
Rhodamine B:
Visualization of lipids
Aniline phthalate:
Visualization of carbohydrates
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Before
After 26

27. Thin Layer and Paper Chromatography
A solution of a mixture is applied as a spot/band at the bottom of the plate and allowed to travel with the solvent up the plate
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28. Thin Layer and Paper Chromatography Relative mobility (Rf) values
The ratio of distance travelled by the component (from origin) compared with distance travelled by the solvent front ( from origin) is called relative mobility (Rf) values
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29. Interpreting the Results
Relative mobility: Rf
The larger the Rf value, the farther the compound traveled up the plate.
An Rf value is a physical property that can be used for identification purposes.
But it does depended on the conditions under which it is measured.
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30. Application of TLC
Used to determine which conditions are ideal to use in column chromatography.
Ex: which solvent system to use
Quick, fast, and inexpensive
It is also used to monitor column chromatography.
Used to quantify the amount of a component present.
Area of the spot
Spot extraction, then measure the amount
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31. Application of TLC Used to determine the purity of a sample.
Can be used to isolate purified substances
It is often used in forensic drug analysis, explosives analysis, toxicology and analysis of dyes and inks.
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32. Column Chromatography
How does Chromatography work?
Chromatography is a method for separating the components of a mixture by different absorption between a stationary phase and a mobile phase (Moving phase).

33. Paper Chromatography

34. Paper Chromatography Stationary phase = cellulose paper
Mobile phase = liquid solvent
The solvent travels up the filter paper via capillary action and carries the sample with it.
The procedure for paper chromatography is exactly like that described for TLC.
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35. Self Test:
Q: What’s wrong with the following chamber setup? A: The beaker is not covered so it’s not saturated with solvent vapor.
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36. Paper Chromatography Experimental Setup
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37. Two Way Paper Chromatography
Aids in separating components that have similar Rf values.
Procedure:
A single spot of sample is placed towards one end of the origin line.
Placed into solvent; it is removed when the solvent line nears the top.
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38. References
Chromatography. URL: aphy. Accessed: June 5, 2007.
Creating a Central Science. URL: pubs.acs.org/hotartcl/tcaw/98 /sep/creat.html. Accessed: June 20, 2007.
Determining solvent systems for TLC and Column Chromatogra- phy. URL: html. Accessed: June 20, 2007.
Paper Chromatography. URL: r_Chromatography. Accessed: June 5, 2007.
Paper Chromatography. URL: lysis/chromatography/paper.html. Accessed: June 20, 2007.
Paper Chromatography: Introduction. URL: peer.tamu.eu/podiu m_poster_presentations/paper%20chromatography%20Handout.doc. Accessed: June 20, 2007.
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39. THANK YOU
for
Your Kind
Attention
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