9. Observe the spots that appear as orange on a yellow background under UV light.
10. Scrap off bands from the plate and treat as preparative TLC
with diethyl ether (Subheading 3.14, steps 11 and 12).
3.16 Location of
Double Bonds in
Purified MAME by
PeriodatePermanganate
Oxidation
Characterization of the resulting fragments indicates the position
and the configuration cis or trans double bonds
1. In a screw-capped tube or Erlenmeyer, transfer dry pure
MAME (Subheading 3.14) (around 1–5 mg).
2. Add 0.6 mL toluene for better solubilization.
3. Add 4 mL ter-butyl alcohol.
4. Add 2.4 mL aqueous 0.002 M sodium carbonate solution.
5. Add 1.2 mL distilled water.
6. Add 2.7 mL stock oxidant solution (Subheading 2.16, item 3).
7. Place the tube at 30
C overnight under constant shaking.
8. Stop the reaction by adding solid sodium metabisulfite until
discoloration.
9. Add a few drops of 20% H 2 SO 4 in water until acidification
(control with pH paper).
10. Vortex and let stand for 5 min.
11. Extract the aqueous phase three times with diethyl ether.
12. Combine the ether phases.
13. Wash the ether phase with 3 ml of distilled water. Repeat three
folds this step until pH of distilled water is monitored with pH
paper (Subheading 3.2, step 7).
14. Dry the ether phase under nitrogen.
15. Add 1 mL CH 2 Cl 2 , 1 mL of CH 3 OH, and 100 μL of
TMS-diazomethane for methylation of the resulting acids (see
Notes 2 and 10b).
16. After 30 min of contact at RT, evaporate the reagents by air or
nitrogen stream under a well-ventilated hood.
17. Analyze the result of the reaction by TLC with dichloromethane as running solvent.
18. Characterize the fragments by GC/MS according to their
retention times and their fragmentation patterns (Fig. 2; see
Note 18).
3.17 Characterization of Hydroxyl
Functions by Mass
Spectrometry After
Silylation
1. Place 1 mg of purified dry “free lipid” (Subheading 3.12) or
MAME (Subheading 3.14) in a screw-capped tube,
under hood.
2. Add eight drops of anhydrous pyridine.
Lipid and Lipoarabinomannan
133
10. Scrap off bands from the plate and treat as preparative TLC
with diethyl ether (Subheading 3.14, steps 11 and 12).
3.16 Location of
Double Bonds in
Purified MAME by
PeriodatePermanganate
Oxidation
Characterization of the resulting fragments indicates the position
and the configuration cis or trans double bonds
1. In a screw-capped tube or Erlenmeyer, transfer dry pure
MAME (Subheading 3.14) (around 1–5 mg).
2. Add 0.6 mL toluene for better solubilization.
3. Add 4 mL ter-butyl alcohol.
4. Add 2.4 mL aqueous 0.002 M sodium carbonate solution.
5. Add 1.2 mL distilled water.
6. Add 2.7 mL stock oxidant solution (Subheading 2.16, item 3).
7. Place the tube at 30
C overnight under constant shaking.
8. Stop the reaction by adding solid sodium metabisulfite until
discoloration.
9. Add a few drops of 20% H 2 SO 4 in water until acidification
(control with pH paper).
10. Vortex and let stand for 5 min.
11. Extract the aqueous phase three times with diethyl ether.
12. Combine the ether phases.
13. Wash the ether phase with 3 ml of distilled water. Repeat three
folds this step until pH of distilled water is monitored with pH
paper (Subheading 3.2, step 7).
14. Dry the ether phase under nitrogen.
15. Add 1 mL CH 2 Cl 2 , 1 mL of CH 3 OH, and 100 μL of
TMS-diazomethane for methylation of the resulting acids (see
Notes 2 and 10b).
16. After 30 min of contact at RT, evaporate the reagents by air or
nitrogen stream under a well-ventilated hood.
17. Analyze the result of the reaction by TLC with dichloromethane as running solvent.
18. Characterize the fragments by GC/MS according to their
retention times and their fragmentation patterns (Fig. 2; see
Note 18).
3.17 Characterization of Hydroxyl
Functions by Mass
Spectrometry After
Silylation
1. Place 1 mg of purified dry “free lipid” (Subheading 3.12) or
MAME (Subheading 3.14) in a screw-capped tube,
under hood.
2. Add eight drops of anhydrous pyridine.
Lipid and Lipoarabinomannan
133