Applications of the Lightscattering Detector

Some examples of the use of the light-scattering detector to monitor the separation of materials that normally require gradient elution for resolution, but are sometimes difficult to sense by other types of detector, are lipids, fatty acids and phospholipids. An example of a chromatogram obtained from a sample containing a mixture of general lipid-class solutes and monitored by the light-scattering detector is shown in Figure 2 (Table 1).

The sample size is rather high for general quantitative liquid chromatographic analyses but the column does not appear to be overloaded. The minimum detectable mass estimated from this chromatogram appears to be about 10 ng of solute. To some extent, this detector provides an alternative to the conventional transport detector as it detects all substances irrespective of their optical or electrical properties. However, modern versions of the conventional wire or ribbon transport detector are reported to have significantly greater sensitivity.

Figure 3 depicts the separation of a mixture of fatty acids. The C18-bonded silica column was 25 cm long, 2.1 mm i.d. and packed with particles of 3 |im diameter. The flow rate was 0.4 mL min"1 and the sol-

Figure 2 The separation of some lipid-class materials monitored by an evaporative light-scattering detector. For key, see Table 1.

vents used were water and acetonitrile. The gradient employed is shown in Table 2 and is typical for a reversed-phase column.

The solutes are initially retained by dispersive forces between the solutes and the stationary phase and are progressively eluted as the dispersive character of the mobile phase is increased with the greater concentration of acetonitrile. The weights quoted appear to be the concentration of each solute in the sample injected 20 | L of solvent. It is seen that an excellent response is obtained and the chromato-gram is quite suitable for accurate quantitative analysis.

The separation of some phospholipids is shown in Figure 4. The column was 10 cm long, 4.6 mm i.d. and packed with particles of silica 3 | m in diameter.

Table 1 Key to Figure 2

Peak

Compound

Mass

Retention time

(M)

(min)

1

Cholesterol ester

5

0.717

2

Triglyceride

18

1.746

3

Cholesterol

10

4.687

4

Unknown

8.860

5

Phosphatidyl choline

10

10.028

6

Phosphatidylethanolamine

10

17.390

Figure 3 The separation of some fatty acids monitored by an evaporative light-scattering detector. Peaks: 1, capric acid (0.10 mg mL~1); 2, lauric acid (0.03 mg mL~1); 3, myristic acid (0.03 mg mL~1); 4, pentadecanoic acid (0.02 mg mL~1); 5, palmitic acid (0.03 mg mL~1).

Figure 4 The separation of some phospholipids monitored by an evaporative light-scattering detector. Peaks: 1, cholesterol (0.15 mg mL~1); 2, palmitic acid (0.25 mg mL~1); 3, phos-phatidylethanolamine (0.15 mg mL~1); 4, phosphatidylserine (0.30 mg mL~1); 5, phosphatidylcholine (0.15 mg mL~1); 6, sphingomyelin (0.15 mg).

Figure 3 The separation of some fatty acids monitored by an evaporative light-scattering detector. Peaks: 1, capric acid (0.10 mg mL~1); 2, lauric acid (0.03 mg mL~1); 3, myristic acid (0.03 mg mL~1); 4, pentadecanoic acid (0.02 mg mL~1); 5, palmitic acid (0.03 mg mL~1).

Figure 4 The separation of some phospholipids monitored by an evaporative light-scattering detector. Peaks: 1, cholesterol (0.15 mg mL~1); 2, palmitic acid (0.25 mg mL~1); 3, phos-phatidylethanolamine (0.15 mg mL~1); 4, phosphatidylserine (0.30 mg mL~1); 5, phosphatidylcholine (0.15 mg mL~1); 6, sphingomyelin (0.15 mg).

The flow rate was i.25mLmin~l and the solvents used were water, isopropanol and w-hexane. The gradient employed is shown in Table 3 and has obviously been specially developed for this type of separation on silica gel.

In this separation the solutes are largely retained by polar forces and are progressively eluted by increasing the proportion of isopropanol and water.

The strong polar solvents deactivate the stationary phase by preferential adsorption and this allows the strong dispersive forces between the solutes and the hexane to elute the solutes. Again, the weights quoted appear to be the concentration of each solute in the sample injected in 20 |L of solvent. It is clear that the detector is quite sensitive to these solutes and, again, the response and resolution are more than adequate for accurate quantitative analysis.

Table 2 Gradient for a typical reversed-phase column with solvents A (water) and B (acetonitrile)

0 min

5min

10 min

20 min

% B 77

80

90

95

Table 3 Gradient using solvents A (isopropanol), B (n-hexane)

and C (water)

0 min

7min

15min

%A

58

52

52

%B

40

40

40

%C

2

8

8

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