Expt 4. TLC-MS Determination of Pigments in Dried Peppers

Relevant textbook reading - Mohrig, Chapter 17

Other References - (1) Elder, J. W.; Abbruzzese, J.; Murray, J.; and Zielski, M. "Separation of paprika pigments. An introductory TLC experiment" J. Chem. Ed. 1976, 53, 43. (2) Wikipedia- Atmospheric pressure chemical ionization, https://en.wikipedia.org/wiki/Atmospheric-pressure_chemical_ionization, accessed March 3, 2017.

Overview - In this lab you will use thin-layer chromatography (TLC) to separate the small organic compounds found in paprika or other dried chili peppers, such as cayenne or red pepper flakes. The separated spots on the TLC plates will then be subjected to mass spectrometry using atmospheric pressure chemical ionization (APCI), which should allow identifications to be made of some of them. APCI-MS differs from the most common type of mass spectrometry, EI-MS (electron-impact ionization) in that the ionization of the molecules occurs via proton transfer reactions to form "M + H" molecular ions. Unlike in EI-MS, these molecular ions have m/z values that equal the MW of the compound plus one. Another major difference is that APCI is a "soft" ionization method, meaning that little fragmentation of the molecular ion is observed, making it easier to be sure about the actual molecular weight of the compound.

Procedures

Extraction

1. Obtain 250 mg of paprika or another form of dried chili pepper (bring your own if you wish!).
2. Extract the sample with 5 mL CH₂Cl₂ by vigorously stirring the mixture for 5-10 min.
3. Filter the solution to remove fine particles by microscale gravity filtration through celite (Mohrig, chap 10.3).

Standard Solutions

Make standard solutions of beta-carotene and capsaicin (two compounds expected to be present in dried peppers) by dissolving 10 mg of each compound in 1.0 mL CH₂Cl₂.

TLC - Refer to Mohrig, Chapter 17, for details of how to carry out these procedures.

1. Obtain a TLC plate (silica gel with fluorescein indicator) and use a straight edged to lightly make a pencil line 1 cm form one end.
2. Make three spots of the pepper extract solution alongside one spot each of the standard solutions. Make sure to record in your notebook the order in which you make these spots.
3. Develop the TLC plate in a TLC-jar using CH₂Cl₂ as the elution solvent.
4. Visualize the plate under UV light in order to detect any colorless spots that might be present. Outline these spots lightly with a pencil.
5. Measure distances of all spots, calculate R_f values and carefully sketch the developed plate in your lab notebook.

TLC-MS

The instructor will assist you in obtaining APCI mass spectra of several of the spots on your TLC plate. The mass spectrometer is located in SLC 339.

Report

Do some literature research in order to find out what compounds (besides capsaicin and beta-carotene) can typically be expected in dried chili peppers. Propose IDs for compounds found in your sample based on (1) R_f value matches to the standard solutions (2) MW and color matches to compounds found in your literature research. How could the peak identifications be further verified (i.e. proven)?