13. The concentration of the preparation can be adjusted since the
response is linear between 2 and 40 ng for all types of mycolic
acids, but for a quantitative analysis, the linearity has to be
checked for each apparatus.
14. Experimenter has to prepare carefully the mobile phase since a
very small variation in water content can have an important
effect on the retention time. The replacement of acetonitrile by
methanol does not improve the separation, so acetonitrile is
used in order to not accentuate viscosity due to isopropanol. A
better resolution can be obtained with 2% of water, but the run
lasts more than 90 min. A run of 45 min with 1% of water is a
good compromise.
15. When large amounts of pure specific lipids are needed, purification by column adsorption chromatography is often
required. Using this method, compounds are separated
according to their relative polarity and the different types of
lipids bound to the solid phase are separated by elution with
solvent mixtures of increasing polarity. Finally fractions of
interest (PDIM, TDM, GroMM) are rechromatographed on
the Florisil column prior to purification which is achieved by
preparative TLC.
A typical chromatography elution for separating the main
lipids on Florisil column would be:
CHCl 3 : apolar lipid, e.g., PDIM, TAG, PPT.
CHCl 3 /CH 3 OH 99:1 (v/v): diacyl glycerol.
CHCl 3 /CH 3 OH 98:2 (v/v): monoacyl glycerol.
CHCl 3 /CH 3 OH 95:5 (v/v): GroMM.
CHCl 3 /CH 3 OH 90:10 (v/v): TDM.
CHCl 3 /CH 3 OH 85:15 (v/v): TAT.
CHCl 3 /CH 3 OH 80:20 (v/v): peptidolipid.
CHCl 3 /CH 3 OH /H 2 O 65/25/4 (v/v/v): phospholipids
(CL, PG, PE, PIM, and OL).
16. The first fractions containing apolar lipids eluted on Florisil
column with CHCl 3 ) are rechromatographied on a Florisil
column with a gradient of less polar solvents such as diethyl
ether in petroleum ether. A classical elution can be: 0%, 10%,
20%, 30%, 50%, and 100% [32]. In these conditions, the PDIM
fractions are isolated nearly pure with 10% diether ether in
petroleum ether.
17. Argentation thin-layer chromatography is based on the property of double-bond π electrons to form a complex with a
metal-ion. Retention by silica is determined by the number
and the configuration of double bonds. For instance, diethylenic MA are more retained than monoethylenic ones. A trans
146
Marie-Antoinette Lane ´ elle et al.
response is linear between 2 and 40 ng for all types of mycolic
acids, but for a quantitative analysis, the linearity has to be
checked for each apparatus.
14. Experimenter has to prepare carefully the mobile phase since a
very small variation in water content can have an important
effect on the retention time. The replacement of acetonitrile by
methanol does not improve the separation, so acetonitrile is
used in order to not accentuate viscosity due to isopropanol. A
better resolution can be obtained with 2% of water, but the run
lasts more than 90 min. A run of 45 min with 1% of water is a
good compromise.
15. When large amounts of pure specific lipids are needed, purification by column adsorption chromatography is often
required. Using this method, compounds are separated
according to their relative polarity and the different types of
lipids bound to the solid phase are separated by elution with
solvent mixtures of increasing polarity. Finally fractions of
interest (PDIM, TDM, GroMM) are rechromatographed on
the Florisil column prior to purification which is achieved by
preparative TLC.
A typical chromatography elution for separating the main
lipids on Florisil column would be:
CHCl 3 : apolar lipid, e.g., PDIM, TAG, PPT.
CHCl 3 /CH 3 OH 99:1 (v/v): diacyl glycerol.
CHCl 3 /CH 3 OH 98:2 (v/v): monoacyl glycerol.
CHCl 3 /CH 3 OH 95:5 (v/v): GroMM.
CHCl 3 /CH 3 OH 90:10 (v/v): TDM.
CHCl 3 /CH 3 OH 85:15 (v/v): TAT.
CHCl 3 /CH 3 OH 80:20 (v/v): peptidolipid.
CHCl 3 /CH 3 OH /H 2 O 65/25/4 (v/v/v): phospholipids
(CL, PG, PE, PIM, and OL).
16. The first fractions containing apolar lipids eluted on Florisil
column with CHCl 3 ) are rechromatographied on a Florisil
column with a gradient of less polar solvents such as diethyl
ether in petroleum ether. A classical elution can be: 0%, 10%,
20%, 30%, 50%, and 100% [32]. In these conditions, the PDIM
fractions are isolated nearly pure with 10% diether ether in
petroleum ether.
17. Argentation thin-layer chromatography is based on the property of double-bond π electrons to form a complex with a
metal-ion. Retention by silica is determined by the number
and the configuration of double bonds. For instance, diethylenic MA are more retained than monoethylenic ones. A trans
146
Marie-Antoinette Lane ´ elle et al.