Lab I: Preparation of Bixin Extract and its Thin Layer Chromatography

Draw the UV region of EM spectrum

• Absorption at UV and visible wavelengths: promotion of e- from HOMO to LUMO

• Extended conjugation reduces HOMO to LUMO gap -> lowers the frequency = absorption at longer wavelengths

What is observed color?

Observed color is complementary to the color of the wavelength absorbed.

Draw the structures

Draw the flow chart

Safety precautions?

• Dichloromethane is a carcinogenic compound.

• Double glove.

• If gloves are damaged:

  • Remove gloves

  • Wash hands for 20 sec w/ soap + warm water

  • Replace w/ new pair of gloves

Waste disposal?

• Annatto seeds —> biohazard waste box

• Buchner funnel filter paper —> biohazard waste box

• Vacuum filter flask, buchner funnel, 50mL beaker rinsed + emptied —> C,H,O Halogenated & acetone rinsings

• TLC capillary tubes —> red sharps container

Properties of chromatography?

• Routinely used as a qualitative analytical tool

• Simple, economical method for separation of components in a mixture.

• Identification of Unknowns by comparison with known reference samples.

• Helps in following the progress of a chemical reaction.

• Helps in determining the effectiveness of a purification.

• Helps to determine the conditions for macroscopic separations (Column Chromatography).

• No restriction on sample type – Organic, Inorganic, Biological or Medical.

• High Sensitivity – Detection of μg amounts or less (10-6 g)

Draw the chromatography stationary phase vs. mobile phase table

What are the 4 types of chromatographic methods?

1. TLC - Thin Layer Chromatography

2. LC – Liquid Chromatography (SP- Silica gel or Alumina; MP – a liquid)

3. HPLC - High Performance Liquid Chromatography (utilizes high pressure exerted by mechanical pumps to force the mobile phase through a very small diameter column packing that contains the stationary phase).

4. GC - Gas Chromatography (MP – a gas)

What are 2 examples of adsorbents?

1. Silica gel (SiO2) - most commonly used, inexpensive stationary phase.

2. Aluminum oxide, Alumina Al2O3

What is silica gel?

• Extended covalent network of tetrahedral Si atoms bridged by “O” atoms – terminating in very polar Silanol (Si-OH) groups, thus creating a very polar surface.

• The presence of these OH groups renders the surface of silica gel highly polar.

What is the composition of a TLC plate?

250 μm silica gel layer impregnated with a fluorescent indicator, on a plastic backing

What are the results of interaction w/ only stationary phase?

• Highly polar molecules will interact strongly with the polar Si-OH bonds in the silica gel adsorbent – resulting in slow movement up the TLC plate.

• Weakly polar molecules are held less tightly than the polar species on the silica gel adsorbent – resulting in quicker movement up the TLC plate

What does adding CaSO4 do?

CaSO4 – added to help silica gel bind to a plate compounds

What is the formula for ratio to the front (Rf) factor?

What are the 4 steps of TLC?

1. Application of Sample: Use a different TLC Capillary tube (open on both ends) to spot the standard solution of each solute.

2. Development of Sample: This is when the separation actually occurs inside the TLC Development Chamber.

3. Visualization of Sample: The TLC plate will be viewed under UV light.

4. Interpretation of Results: Comparison of Retention factors

Sample application reminders?

• Draw baseline 1.5 cm from bottom of plate.

• Spot lightly on ROUGH SIDE of TLC plate (avoid breaking silica gel).

• Draw solvent front ~1 cm from top of plate.

Draw the schematic for the developing chamber to run a TLC plate