4. Water (Milli-Q grade).
5. HPLC-grade solvents: Acetonitrile, methanol, isopropanol,
and cyclohexane (see Note 2).
6. Formic acid.
7. 90% isopropanol, 10% acetonitrile, 0.1% formic acid (v/v/v)
(base mix for sample).
8. 90% isopropanol/1% water/0.1% formic acid (v/v/v)
(solvent A).
9. Acetonitrile/water 99/1% water (v/v) (solvent B).
10. 1.5-mL vials with screw caps N9 (Macherey-Nagel) and glass
inserts.
11. Synthetic corynomycolic acid C32 (gift of Drs Asselineau) can
be used if an internal standard IST is required.
12. Automatic pipettes (2.5, 10, 200 μL).
2.8 Enrichment of
Lipid Extract in PDIM,
PGL, and GPL by Mild
Alkaline Hydrolysis
1. 0.2 M NaOH in methanol.
2. Glacial acetic acid.
3. HPLC-grade solvents: Chloroform and methanol (see Note 2).
4. Pasteur pipettes.
5. Glass test tubes.
6. Incubator at 37
C.
7. Air or nitrogen stream.
2.9 Enrichment of
Lipid Extract in LongChain Compounds
(TDM, TMM, GroMM, or
MAME) by Methanol
Precipitation (See
Note 4)
1. Refrigerated centrifuge.
2. Glass conic centrifuge tube (Corex quality).
3. HPLC-grade solvents: Chloroform and methanol (see Note 2).
4. Pasteur pipettes.
5. Glass test tubes.
6. Air or nitrogen stream.
2.10 Enrichment of
Lipid Extract in
Phospholipids and
Sulfolipids
1. Refrigerated centrifuge.
2. Glass conic centrifuge tube (Corex quality).
3. HPLC-grade solvents: Chloroform and acetone (see Note 2).
4. Pasteur pipettes.
5. Glass test tubes.
2.11 Purification of
Defined Lipids by
Adsorption
Chromatography on
Florisil Column
1. A glass column equipped with cotton at the bottom, a Teflon
stopcock, and a reservoir at the top to receive the solvent.
2. Round-bottom flasks.
3. A rotary vacuum evaporator (see Note 3).
Lipid and Lipoarabinomannan
119
5. HPLC-grade solvents: Acetonitrile, methanol, isopropanol,
and cyclohexane (see Note 2).
6. Formic acid.
7. 90% isopropanol, 10% acetonitrile, 0.1% formic acid (v/v/v)
(base mix for sample).
8. 90% isopropanol/1% water/0.1% formic acid (v/v/v)
(solvent A).
9. Acetonitrile/water 99/1% water (v/v) (solvent B).
10. 1.5-mL vials with screw caps N9 (Macherey-Nagel) and glass
inserts.
11. Synthetic corynomycolic acid C32 (gift of Drs Asselineau) can
be used if an internal standard IST is required.
12. Automatic pipettes (2.5, 10, 200 μL).
2.8 Enrichment of
Lipid Extract in PDIM,
PGL, and GPL by Mild
Alkaline Hydrolysis
1. 0.2 M NaOH in methanol.
2. Glacial acetic acid.
3. HPLC-grade solvents: Chloroform and methanol (see Note 2).
4. Pasteur pipettes.
5. Glass test tubes.
6. Incubator at 37
C.
7. Air or nitrogen stream.
2.9 Enrichment of
Lipid Extract in LongChain Compounds
(TDM, TMM, GroMM, or
MAME) by Methanol
Precipitation (See
Note 4)
1. Refrigerated centrifuge.
2. Glass conic centrifuge tube (Corex quality).
3. HPLC-grade solvents: Chloroform and methanol (see Note 2).
4. Pasteur pipettes.
5. Glass test tubes.
6. Air or nitrogen stream.
2.10 Enrichment of
Lipid Extract in
Phospholipids and
Sulfolipids
1. Refrigerated centrifuge.
2. Glass conic centrifuge tube (Corex quality).
3. HPLC-grade solvents: Chloroform and acetone (see Note 2).
4. Pasteur pipettes.
5. Glass test tubes.
2.11 Purification of
Defined Lipids by
Adsorption
Chromatography on
Florisil Column
1. A glass column equipped with cotton at the bottom, a Teflon
stopcock, and a reservoir at the top to receive the solvent.
2. Round-bottom flasks.
3. A rotary vacuum evaporator (see Note 3).
Lipid and Lipoarabinomannan
119