4. APPARATUS

   1. High performance liquid chromatograph, consisting of an HPLC pump and an injection valve with 10 /il sample loop.

   2. UV-detector system to measure absorbance at 206 nm.

   3. Column: stainless steel tube, length 200 mm and internal diameter about 4.6 mm, packed with microparticulate spherical silica (5 /i) [Note 1].

   4. Recorder and/or integrator.

   5. Syringe, 25 ij.1 graduated in 1 ill

   6. Flasks, volumetric, 10, ml, 500 ml and 1000 ml

5. Reagents

   1. n-Hexane, HPLC grade

   2. 2-Propanol, HPLC grade

   3. Water, double distilled, filtered through a 0.45 ^ pore size membrane filter.

   4. Sodium acetate, 0.2M. Weigh 27.22 g sodium acetate [3H₂0], analytical grade, into a 1000 ml flask (4.6) and make up to volume with water. Mix.

   5. Acetic acid, 0.2M. Weigh 6.006 g glacial acetic acid, analytical grade, into a 500 ml flask (4.6) and make up to volume with water. Mix.

   6. Acetate buffer (Walpole), pH 4.2. Prepare by mixing 2.65 ml sodium acetate solution (5.4) and 7.35 ml acetic acid solution (5.5).

   7. HPLC mobile phase. Prepare by mixing n-hexane (4.10), 2- propanol (4.2), and acetate buffer (5.6) in the proportion 8:8:1 (V/V).

   8. Reference phospholipids: phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylcholine (PC).

6. PROCEDURE

1. Stabilization of the column

To achieve a stable operating performance of the column it must be charged with a suitable quantity of buffer. This is carried out by passing mobile phase (5.7) through the column for at least 3 days at a flow rate of 0.2 ml/min until a stable baseline is obtained. A stable baseline will result in the retention times of peaks also being stable.

2. HPLC chromatography

Use the mobile phase (5.7) at a flow rate of 2 ml/min.

3. Calibration curve

Accurately weigh 10, 20, 30 and 40 mg quantities of each reference phospholipid (5.8) into separate 10 ml flasks (4.6). Dissolve the phospholipid in about 3 ml mobile phase (5.7), make up to volume with the same solvent and mix well [Note 2].

Construct a calibration curve for each phospholipid by injecting exactly 10 of each of the calibration solutions, recording the peak areas and plotting the latter against the corresponding quantity of phospholipid.

4. Sample analysis

Weigh accurately 50 to 250 mg of the lecithin sample into a small beaker and dissolve in about 3 ml mobile phase (5.7). Transfer quantitatively to a 10 ml flask (4.6), make up to volume with the same solvent, and mix well. In the case of some lecithins a sediment may form after some time. Inject 10 y.1 of the upper layer. The sediment has no influence on the analysis but can affect the column's performance [Note 3].

Record the individual peak areas of the eluted phospholipids, identifying them from the chromatogram (Fig. 1) or by comparison with the retention times observed during the chromatography (6.3) of the reference phospholipids (5.8).

7. Calculation and expression of results

1. Read off from the calibration curves the amount of phospholipids in the injected volume of the solution of the lecithin sample (6.4).

2. The phospholipid content (P), expressed as per cent (m/m), is given by the formula:

where

^{A x 100} A is the amount of phospholipid, in mg,

^{p =} read from the calibration curve (7.1)

m

m is the mass of sample weight, in mg (6.4)

Fig. 1 Chromatograms of soybean phospholipids