l
For the separation of the PIM family of M. tuberculosis and
M. bovis BCG differing by their acylation degrees, i.e., the
four acyl forms of each family of PIM (the dimannoside
PIM 2 and the hexamannoside PIM 6 ) are separated by running the plate with chloroform/methanol/water 60:35:8
(v/v/v) [27].
9. According to the nature of the lipids to be detected, specific
reagents should be used. The primulin reagent reveals all types
of lipid and possesses the great advantage to be convenient for
both analytical and preparative TLC, as it does not degrade
compounds upon reaction. It also allows the visualization of
lipids under UV at 356 nm.
l
For TAG and fatty acid methyl esters (FAME) or mycolic
acid methyl esters (MAME) resulting from the saponification of complex lipids: rhodamine B (0.1% solution in ethanol, diluted to 0.01% in 0.25 M NaH 2 PO 4 solution), a
non-degradative reagent used for preparative TLC, and
visualization under UV light; or molybdophosphoric acid
(10% in ethanol) and charring.
l
For phospholipids: Dittmer-Lester reagent [48].
l
For glycolipids: 0.2% anthrone solution in sulfuric acid,
followed by charring. Observe the blue spots appearing
while charring; note the differences in the colors of glycolipids depending on the nature of the saccharides: intense
blue for PGL or GPL, and first detected glycolipids, bluegreen for PIMs, and blue-gray for trehalose esters
including SGL.
l
For lipids containing free NH 2 (e.g., PE and OL): 0.2%
ninhydrine solution in acetone. After a light charring,
observe the violet spots appearing. It is also possible to
localize phospholipids on the same TLC by cooling the
plate after revelation with ninhydrine at room temperature,
and then spray with Dittmer-Lester reagent [48].
l
OL in M. canetti (migrates as PE): ninhydrine [66]. Peptidolipids from M. fortuitum, M. paratuberculosis or Nocardia cannot be detected by ninhydrine on TLC as they are
devoid of free amino groups [8].
10. (a) Saponification.
Alkaline conditions are the best method for isolating free
acids from complex lipids. Acid treatment is known to cause
the aperture of epoxy ring [11, 12]. Moreover, the conditions
exposed here allow the cleavage of functions resistant to saponification as wax-ester. An observation of the TLC pattern of
mycolates of strains containing keto-mycolates and wax-ester
mycolates of the Mycobacterium avium-intracellulare-scrofulaceum complex, obtained by saponification with
144
Marie-Antoinette Lane ´ elle et al.
For the separation of the PIM family of M. tuberculosis and
M. bovis BCG differing by their acylation degrees, i.e., the
four acyl forms of each family of PIM (the dimannoside
PIM 2 and the hexamannoside PIM 6 ) are separated by running the plate with chloroform/methanol/water 60:35:8
(v/v/v) [27].
9. According to the nature of the lipids to be detected, specific
reagents should be used. The primulin reagent reveals all types
of lipid and possesses the great advantage to be convenient for
both analytical and preparative TLC, as it does not degrade
compounds upon reaction. It also allows the visualization of
lipids under UV at 356 nm.
l
For TAG and fatty acid methyl esters (FAME) or mycolic
acid methyl esters (MAME) resulting from the saponification of complex lipids: rhodamine B (0.1% solution in ethanol, diluted to 0.01% in 0.25 M NaH 2 PO 4 solution), a
non-degradative reagent used for preparative TLC, and
visualization under UV light; or molybdophosphoric acid
(10% in ethanol) and charring.
l
For phospholipids: Dittmer-Lester reagent [48].
l
For glycolipids: 0.2% anthrone solution in sulfuric acid,
followed by charring. Observe the blue spots appearing
while charring; note the differences in the colors of glycolipids depending on the nature of the saccharides: intense
blue for PGL or GPL, and first detected glycolipids, bluegreen for PIMs, and blue-gray for trehalose esters
including SGL.
l
For lipids containing free NH 2 (e.g., PE and OL): 0.2%
ninhydrine solution in acetone. After a light charring,
observe the violet spots appearing. It is also possible to
localize phospholipids on the same TLC by cooling the
plate after revelation with ninhydrine at room temperature,
and then spray with Dittmer-Lester reagent [48].
l
OL in M. canetti (migrates as PE): ninhydrine [66]. Peptidolipids from M. fortuitum, M. paratuberculosis or Nocardia cannot be detected by ninhydrine on TLC as they are
devoid of free amino groups [8].
10. (a) Saponification.
Alkaline conditions are the best method for isolating free
acids from complex lipids. Acid treatment is known to cause
the aperture of epoxy ring [11, 12]. Moreover, the conditions
exposed here allow the cleavage of functions resistant to saponification as wax-ester. An observation of the TLC pattern of
mycolates of strains containing keto-mycolates and wax-ester
mycolates of the Mycobacterium avium-intracellulare-scrofulaceum complex, obtained by saponification with
144
Marie-Antoinette Lane ´ elle et al.