4. Reveal the two non-covered sides of the TLC plate by carefully
spraying with the appropriate reagent (see Note 9).
5. Note the reagent-positive regions.
6. Scrap off the corresponding unrevealed bands with a spatula.
7. Collect the powder in a test tube.
8. Add 3 mL of the solvent used for the migration.
9. Filter the solvent extract containing the purified lipid in a
pre-weighed test tube through a 1-cm Florisil Pasteur pipette
column in order to eliminate silica powder.
10. Dry under air or nitrogen stream the organic phase.
3.13 Purification of
Nonpolar Lipids
(PDIM-A, PDIM-B, and
TAG) by PuriFlash
System
Crude lipid fractions or those enriched in PDIM-A, PDIM-B, and
TAG eluted by CHCl 3 on Florisil column (see Note 16) are purified
on silica gel PuriFlash system (Subheading 2.13).
1. Fill solvent bottles with A: Petroleum ether, B: Petroleum
ether/diethyl ether 9:1 (v/v), C: Chloroform, and D:
Methanol.
2. Solubilize about 50 mg of total lipid extract in 3 mL of petroleum ether.
3. Install the PuriFlash silica gel column (see Subheading 2.13,
item 1).
4. Wash the column with solvents D, C, B, and A, successively
(about 30 mL each).
5. Apply the following method:
l
Injection: Equilibration 4 column volumes (CV), mode
valve injection + pause, injection loop 5 mL.
l
Elution at 20 mL/min:
A
B
C
D
20 CV
100%
10 CV
90%
10%
23 CV
100
25 CV
100
28 CV
50
50
6. Collect (either manually or with a collector programmed) 2 mL
fractions.
7. Concentrate fractions 15–20 and 24–35 separately.
8. Solubilize them in approximatively 0.3 mL CHCl 3 .
9. Spot aliquots with Pasteur pipettes on silica gel G60 0.25-mm
plates.
Lipid and Lipoarabinomannan
131
spraying with the appropriate reagent (see Note 9).
5. Note the reagent-positive regions.
6. Scrap off the corresponding unrevealed bands with a spatula.
7. Collect the powder in a test tube.
8. Add 3 mL of the solvent used for the migration.
9. Filter the solvent extract containing the purified lipid in a
pre-weighed test tube through a 1-cm Florisil Pasteur pipette
column in order to eliminate silica powder.
10. Dry under air or nitrogen stream the organic phase.
3.13 Purification of
Nonpolar Lipids
(PDIM-A, PDIM-B, and
TAG) by PuriFlash
System
Crude lipid fractions or those enriched in PDIM-A, PDIM-B, and
TAG eluted by CHCl 3 on Florisil column (see Note 16) are purified
on silica gel PuriFlash system (Subheading 2.13).
1. Fill solvent bottles with A: Petroleum ether, B: Petroleum
ether/diethyl ether 9:1 (v/v), C: Chloroform, and D:
Methanol.
2. Solubilize about 50 mg of total lipid extract in 3 mL of petroleum ether.
3. Install the PuriFlash silica gel column (see Subheading 2.13,
item 1).
4. Wash the column with solvents D, C, B, and A, successively
(about 30 mL each).
5. Apply the following method:
l
Injection: Equilibration 4 column volumes (CV), mode
valve injection + pause, injection loop 5 mL.
l
Elution at 20 mL/min:
A
B
C
D
20 CV
100%
10 CV
90%
10%
23 CV
100
25 CV
100
28 CV
50
50
6. Collect (either manually or with a collector programmed) 2 mL
fractions.
7. Concentrate fractions 15–20 and 24–35 separately.
8. Solubilize them in approximatively 0.3 mL CHCl 3 .
9. Spot aliquots with Pasteur pipettes on silica gel G60 0.25-mm
plates.
Lipid and Lipoarabinomannan
131