4. Pour the slurry with gently shaking to eliminate bubbles.
5. Rinse with one volume of the solvent.
6. Allow the solvent to run slowly (by drops) through the column.
7. Dissolve the “free” lipid extract (Subheading 3.1, step 17) in
the minimum amount (few mL) of the most apolar
solvent used.
8. With the solvent level at the top of the adsorbent, add carefully
the lipid suspension on the top of the column and let it to
adsorb.
9. Proceed to the elution gradient (volume 30–50 mL) (see Note
15).
10. Change the round bottom flask after each elution.
11. Evaporate the solvent under vacuum with a rotary evaporator
(see Note 3).
12. Dissolve the dried lipid in a minimum of chloroform.
13. Transfer the material in pre-weighed test tubes.
14. Evaporate the solvent under air or nitrogen stream.
15. Dissolve dried lipids in chloroform at an approximate concentration of 10 mg/mL.
16. Spot 50 μL of the fraction on plates with a Pasteur pipette or a
syringe and follow the separation by TLC with appropriate
solvents (see Note 8).
17. Stain the plate with a spray adapted to the nature of compounds (see Note 9).
18. Pool fractions exhibiting similar lipid contents.
19. Dry under air or nitrogen stream.
20. Re-chromatograph the pooled fraction on Florisil column
(repeat steps 8–17) by adapting the solvent elution to the
structure of the lipid (see Note 16).
3.12 Final
Purification of PDIM,
TDM, and GroM by
Preparative TLC
1. Spot the sample, i.e., column chromatography fractions (see
Note 15) up to 10 mg in chloroform for 20 Â 20 cm plate,
with a Pasteur pipette or a 100 μL glass syringe as a line at the
origin of the TLC plate.
2. Run the plate in the TLC tank using appropriate solvents, e.g.,
petroleum ether/diethyl ether 9:1 (v/v) for PDIM and TAG,
chloroform/methanol 9:1 (v/v) for GroMM and TDM.
3. After development, first protect the whole TLC surface with a
glass plate, except a small band on each vertical side to be
visualized as a control for the detection of lipid bands by
spraying.
130
Marie-Antoinette Lane ´ elle et al.
5. Rinse with one volume of the solvent.
6. Allow the solvent to run slowly (by drops) through the column.
7. Dissolve the “free” lipid extract (Subheading 3.1, step 17) in
the minimum amount (few mL) of the most apolar
solvent used.
8. With the solvent level at the top of the adsorbent, add carefully
the lipid suspension on the top of the column and let it to
adsorb.
9. Proceed to the elution gradient (volume 30–50 mL) (see Note
15).
10. Change the round bottom flask after each elution.
11. Evaporate the solvent under vacuum with a rotary evaporator
(see Note 3).
12. Dissolve the dried lipid in a minimum of chloroform.
13. Transfer the material in pre-weighed test tubes.
14. Evaporate the solvent under air or nitrogen stream.
15. Dissolve dried lipids in chloroform at an approximate concentration of 10 mg/mL.
16. Spot 50 μL of the fraction on plates with a Pasteur pipette or a
syringe and follow the separation by TLC with appropriate
solvents (see Note 8).
17. Stain the plate with a spray adapted to the nature of compounds (see Note 9).
18. Pool fractions exhibiting similar lipid contents.
19. Dry under air or nitrogen stream.
20. Re-chromatograph the pooled fraction on Florisil column
(repeat steps 8–17) by adapting the solvent elution to the
structure of the lipid (see Note 16).
3.12 Final
Purification of PDIM,
TDM, and GroM by
Preparative TLC
1. Spot the sample, i.e., column chromatography fractions (see
Note 15) up to 10 mg in chloroform for 20 Â 20 cm plate,
with a Pasteur pipette or a 100 μL glass syringe as a line at the
origin of the TLC plate.
2. Run the plate in the TLC tank using appropriate solvents, e.g.,
petroleum ether/diethyl ether 9:1 (v/v) for PDIM and TAG,
chloroform/methanol 9:1 (v/v) for GroMM and TDM.
3. After development, first protect the whole TLC surface with a
glass plate, except a small band on each vertical side to be
visualized as a control for the detection of lipid bands by
spraying.
130
Marie-Antoinette Lane ´ elle et al.