2. Heat at 110
C for 3 h in a screw-capped glass tube
(18 Â 1.5 cm).
3. After cooling, acidify for 10 min the mixture with 1 mL of 20%
H 2 SO 4 solution to release free FA from salts.
4. Check the pH (pH 1) with pH paper.
5. Add 3 mL water and 3 mL diethyl ether.
6. Vortex.
7. Transfer the upper diethyl ether phase in a fresh test tube
(18 Â 1.5 cm is recommended for an easy extraction).
8. Add 3 mL of ether to the aqueous phase.
9. Vortex.
10. Transfer the upper diethyl ether in the test tube of step 8.
11. Repeat step 9 twice again with the remaining lower phase.
12. Pool the diethyl ether phases from steps 8 and 9.
13. Wash the ether phase with 1 mL of distilled water, four or five
times (until neutrality, i.e., pH of distilled water, monitored
with a pH paper).
14. Dry the diethyl ether phase under air or nitrogen stream. This
represents total “free fatty acids.”
15. Add 1 mL dichloromethane, 1 mL of methanol, and 100 μL of
TMS-diazomethane (see Notes 2 and 10b).
16. Evaporate the reagent by air or nitrogen stream in a wellventilated hood, after 30 min of contact at RT. This represents
total methyl esters, i.e., FAME and MAME.
3.4 Characterization
of MAME Profile by 1-D
and 2-D TLC (One- and
Two- Dimension TLC)
1. Dissolve FAME and MAME (from Subheading 3.3, step 10) in
dichloromethane at an approximate concentration 10 mg/mL.
2. Spot around 50 μL on the TLC plate, only one spot in a corner
for two-dimension TLC (2-D TLC).
3. Run the one-dimension TLC (1-D TLC) plate in dichloromethane or in 10% diethyl ether in petroleum ether (see Note
11). Dry the plates at room temperature between each run.
4. Run the two-dimension TLC (2-D TLC) first in 5% acetone in
petroleum ether, three times, dry the plate, turn the plate, and
run it in the second direction in 3% acetone in toluene (see
Note 11). Dry the plates at room temperature between
each run.
5. Stain the TLC plate by spraying a 10% solution in ethanol of
molybdophosphoric acid and charring or rhodamine B and
observation under UV light.
6. An analytical TLC of MAME from representative mycobacteria
is shown in Fig. 3A, B. The two systems of solvents allow the
separation of MAME.
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Marie-Antoinette Lane ´ elle et al.
C for 3 h in a screw-capped glass tube
(18 Â 1.5 cm).
3. After cooling, acidify for 10 min the mixture with 1 mL of 20%
H 2 SO 4 solution to release free FA from salts.
4. Check the pH (pH 1) with pH paper.
5. Add 3 mL water and 3 mL diethyl ether.
6. Vortex.
7. Transfer the upper diethyl ether phase in a fresh test tube
(18 Â 1.5 cm is recommended for an easy extraction).
8. Add 3 mL of ether to the aqueous phase.
9. Vortex.
10. Transfer the upper diethyl ether in the test tube of step 8.
11. Repeat step 9 twice again with the remaining lower phase.
12. Pool the diethyl ether phases from steps 8 and 9.
13. Wash the ether phase with 1 mL of distilled water, four or five
times (until neutrality, i.e., pH of distilled water, monitored
with a pH paper).
14. Dry the diethyl ether phase under air or nitrogen stream. This
represents total “free fatty acids.”
15. Add 1 mL dichloromethane, 1 mL of methanol, and 100 μL of
TMS-diazomethane (see Notes 2 and 10b).
16. Evaporate the reagent by air or nitrogen stream in a wellventilated hood, after 30 min of contact at RT. This represents
total methyl esters, i.e., FAME and MAME.
3.4 Characterization
of MAME Profile by 1-D
and 2-D TLC (One- and
Two- Dimension TLC)
1. Dissolve FAME and MAME (from Subheading 3.3, step 10) in
dichloromethane at an approximate concentration 10 mg/mL.
2. Spot around 50 μL on the TLC plate, only one spot in a corner
for two-dimension TLC (2-D TLC).
3. Run the one-dimension TLC (1-D TLC) plate in dichloromethane or in 10% diethyl ether in petroleum ether (see Note
11). Dry the plates at room temperature between each run.
4. Run the two-dimension TLC (2-D TLC) first in 5% acetone in
petroleum ether, three times, dry the plate, turn the plate, and
run it in the second direction in 3% acetone in toluene (see
Note 11). Dry the plates at room temperature between
each run.
5. Stain the TLC plate by spraying a 10% solution in ethanol of
molybdophosphoric acid and charring or rhodamine B and
observation under UV light.
6. An analytical TLC of MAME from representative mycobacteria
is shown in Fig. 3A, B. The two systems of solvents allow the
separation of MAME.
126
Marie-Antoinette Lane ´ elle et al.