Cardiolipin

Column chromatography
The cardiolipin phospholipids were dissolved in 30 ml of eluent (2-propanol- cyclohexane-water 50:43:7 (v/v/v)) and isocratically eluted over a 135 x 3 cm column, packed with Polygosil
60-63100 (Marchery-Nagel and Co.), with a flow rate of
10 ml/min. The column was packed and equilibrated in the same eluent system. The eluate was collected in fractions of about 200 ml; the content of lipid was determined by HPTLC. Cardiolipin was the first component in the eluate. Fractions 5-10, containing cardiolipin and some contaminants with a high Rf value, were collected. Following fractions, containing other acidic phospholipids, were discarded. The yield was about 2.5 g.
HPLC
About 300 mg of the cardiolipin was dissolved in 3 ml of eluent (2-propanol-cyclohexanewater
45:50:5 (v/v/v)) and eluted over a 250 X 22 mm
HPLC column, packed with Lichrosorb Si 60-5 (Merck), with a flow rate of about 17 ml/min. The eluate was detected with a Melz differential refractometer (LCD 201).
The fractions that contain pure cardiolipin (elution time:
10-20 min) were pooled and evaporated to dryness. After each elution the column material was rinsed with 500 ml of methanol and re-equilibrated with the eluent until the refractive index of the eluate was constant. The total yield was 1.5-2.1 g.
Conversion of cardiolipin into the sodium salt form
During the chromatographic steps, a small part of the cardiolipin was converted from the calcium salt form into other undefined salt forms (see Table 1). That is why it was considered necessary that the HPLC-purified cardiolipin was first converted quantitatively into the calcium salt, in order to enable a more efficient and quantitative conversion into the sodium salt.
The HPLC-purified cardiolipin (2 g) was dissolved in
100 ml of chloroform, whereafter 200 ml of methanol and
100 ml of 0.1 M CaClZ were added. The mixture was shaken for 5 min and phase separation was induced by adding 50 ml of