1. A study about a separation technique
Chromatography Lab
A study about a separation technique

2. What’s involved? Phases of Chromatograpy:
Mobile phase – a solvent compatible with the mixture’s solutes – moves the mixture for separation
Stationary phase – material that holds the mixture and does the separating – sets the criteria used for molecule separation
Discussion on the Analogy of a strainer when making a mixture of different size pasta noodles
Discussion of gel electrophoresis

3. Chemistry Background
Mixture – combo of substances which can be separated
Solution – type of mixture
#1 chlorophyll pigment #2 carotenoid pigment
Pigment molecules in each – Are the solute molecules in the solution
Liquid organic compound – Is the solvent molecules
Solvents dissolve solutes
Remember, molecules interact with each other – intermolecular forces

4. How does it separate? The picture on the right is has an unclear: the
arrow pointing to the liquid in the vial is the
solvent. I think the author is saying a “solvent
mixutre” Where would the mixture be?

5. Materials Needed: Two glass vials with lids per pair
One bottle of developing solution and an eyedropper to share
Two bottle of plant pigments: chlorophyll and carotenoid to share
Vial of capillary tubes to apply on silica gel strip to share
Pair does two chromatography separations: one does chlorophyll and one does carotenoid

6. Removing the Chromatograph
Open the vial
Look for the line that marks the wet and the dry area.
Place it on the white paper. Mark the solvent front similar to the illustration.
Mark origin.
Id bands and label

7. Assignment during the lab
Read AP Lab 4A and complete lab reading questions. This lab discusses background on how paper chromatography works. Remember, your lab is using thin layer chromatography. Both have a stationery, mobile and mixture involved and follow the same guiding principles for separation.
Draw a table on another half sheet of white unlined paper with a ruler. This will be used to display band #, color and Rf value

8. Data Organization Band # Color Rf Value Solvent front 1 2 3 4 5 6 7 8
Table drawn below chromatograph
origin
Chromatograph taped to the half sheet of white paper

9. Details of Chromatography

10. How does thin layer chromatography work?
The stationary phase - silica gel
Silica gel is a form of silicon dioxide (silica). The silicon atoms are joined via oxygen atoms in a giant covalent structure. However, at the surface of the silica gel, the silicon atoms are attached to -OH groups.
So, at the surface of the silica gel you have Si-O-H bonds instead of Si-O-Si bonds. The diagram shows a small part of the silica surface.
The surface of the silica gel is very polar and, because of the -OH groups, can form hydrogen bonds with suitable compounds around it as well as van der Waals dispersion forces and dipole-dipole attractions
The other commonly used stationary phase is alumina - aluminium oxide. The aluminium atoms on the surface of this also have -OH groups attached. Anything we say about silica gel therefore applies equally to alumina.

11. Are bands of the same color the same pigment? Explain

12. How To Quantify Data with the Rf Value
Watch the video to understand how to calculate the Rf value. Click on the above link.

13. Calculating Rf

14. Be ready for an evaluation
Lab Assignment:
A Data Sheet which has the following:
A title for the chromatography
An annotated chromatograph – origin, solvent front, labeled bands
A complete data table with Band#, color, Rf value
Name, Period and Date in an appropriate area on paper
Review background, procedure and application info
Be ready for an evaluation
Even if the bands on the chromatograph share the same color to the naked eye, why are they not considered the same compound? Explain.

15. Further Your Understanding
Various Uses for Thin Layer Chromatography

16. Column Chromatography
Size Exclusion Lab: separating a mixture of biomolecules

17. The Biomolecules
Hemoglobin
B12
Look for the Heme group

18. Separation of Hemoglobin and B12 Molecules

19. What do the numbers tell you about the separation process?
What does a colorless liquid in a fraction tube indicate?
What would a fractions look like with a poor separation technique?

20. What is wrong with the setup?

21. What is wrong with these separations?

22. Other Types of Column Chromatography
Ion Exchange
Affinity - this type of chromatography is used in molecular biology when you want to separate a protein of interest from a mixture. Sometimes a researcher will have a cell produce a protein but the cell doesn’t just produce the protein of interest. It produces many others. So, the column will contain beads which have an antibody which the protein of interest binds to. The relationship between antibody and protein is specific. Recall, the Holly Gets Sick Video from Honors Biology. Your body makes specific antibodies to a specific antigen. This idea is the foundation for affinity chromatography.