Monocarboxylic and Dicarboxylic Tetrapyrroles with Diethyl Ether

1. Prepare a HEAR fraction hypophase as above and transfer to a conical centrifuge tube.

2. Transfer monocarboxylic tetrapyrroles to diethyl ether by adding:

1/5 volume of diethyl ether, 1/17 volume of saturated NaCl, 1/70 volume of 0.37 MKH2PO4 at pH 7.7.

3. Mix thoroughly and resolve the phases by centrifugation at 1500x g for 30 seconds at room temperature.

4. Remove the ether phase using a Pasteur pipet.

5. Re-extract the HEAR fraction 4 to 5 times with small volumes of diethyl ether using centrifugation to resolve the emulsion.

6. Combine the ether extracts for further

Figure 2. Structure of protoporphyrin IX, which is the last common intermediate of chlorophyll and heme.

manipulation of monocarboxylic tetra-pyrroles.

7. For extraction of dicarboxylic tetra-pyrroles from the remaining ether-extracted HEAR fraction, adjust the pH to 4.0 to protonate the free car-boxylic groups.

8. Extract 5 times with small volumes of diethyl ether using centrifugation to breakdown the emulsion.

9. Wash the combined ether extracts containing dicarboxylic tetrapyrroles by passing through a 0.5 M solution of

KH2PO4 adjusted to pH 4.8. This washing step is extremely important for the recording of sharp 77 K spectra in diethyl ether. It is best achieved by removing the diethyl ether extract with a Pasteur pipet and slowly releasing it at the bottom of a 15-mL test tube filled with the phosphate buffer. As the diethyl ether bubbles rise to the top, the aqueous contaminants pass into the buffer. The washed diethyl ether extract can then be removed using a Pasteur pipet.

Figure 3. Mg-protoporphryin IX and its derivatives

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