TLC Solvent Systems – Lipid Migration
CHLOROFORM : METHANOL : WATER (65:25:4)
Purpose: General separation of phospholipids by headgroup polarity.
Notes: Phosphatidylserine, phosphatidylcholine, phosphatidylinositol, co-migrate in this solvent system. Phosphatidylglycerol and cardiolipin co-migrate in this solvent system.
CHLOROFORM : METHANOL : AMMONIUM HYDROXIDE (65:25:4)
Purpose: General separation of phospholipids by headgroup polarity and charge.
Notes: Phosphatidylserine, phosphatidic acid, and phosphatidylinositol do not migrate above the origin in this solvent system. Phosphatidylglycerol and cardiolipin co-migrate in this solvent system.
CHLOROFORM : HEXANE : METHANOL : ACETIC ACID (50:30:10:5)
Purpose: Separation of cardiolipin and phosphatidylglycerol.
TOLUENE : PYRIDINE : WATER (60:60:10)
Purpose: Separation of phosphatidylcholine, phosphatidyl(N,N-dimethylethanolamine), phosphatidyl(N- monomethylethanolamine), phosphatidylethanolamine.
CYCLOHEXANE : ETHYL ACETATE (3:2)
Purpose: Separation of neutral lipids (fatty acids, triglycerides, diglycerides, monoglycerides)
TOLUENE : CHLOROFORM : METHANOL (85:15:5)
Purpose: Separation of diglycerides (1,2 from 1,3 isomer).
TOLUENE : METHANOL (7:3)
Purpose: Separation of ceramide from other sphingolipids.
Notes: Rf of sphingolipids: Ceramide=0.57; Sphingosine=0.14; Sphingomyelin=0.04
SOLVENT SYSTEM CHARTS
| Lipids | Solvent System (Rf) [see chart for system] | |||||
|---|---|---|---|---|---|---|
| i | ii | iii | iv | v | vi | |
| Triglycerides | 0.70 | 0.96 | ||||
| Free Fatty Acid | 0.51 | 0.16 | ||||
| Diglycerides 1,2 | 0.70 | 0.24 | ||||
| Diglycerides 1,3 | 0.32 | |||||
| Monoglycerides | ||||||
| Phosphatidylethanolamine | 0.79 | 0.55 | 0.43 | |||
| Phosphatidyl (Monomethylethanolamine) | 0.71 | 0.41 | 0.33 | |||
| Cardiolipin | 0.67 | 0.56 | 0.38 | |||
| Phosphatidylglycerol | 0.60 | 0.50 | 0.31 | 0.36 | ||
| Phosphatidyl (Dimethylethanolamine) | 0.58 | 0.56 | 0.27 | |||
| Phosphatidic Acid | 0.55 | 0.05 | 0.58 | |||
| Phosphatidylinositol | 0.39 | 0.10 | ||||
| Phosphatidylcholine | 0.34 | 0.30 | 0.17 | |||
| Phosphatidylserine | 0.33 | 0.12 | ||||
| Cerebrosides | 0.94 | 0.55 | ||||
| Ceramides | sf* | sf* | ||||
| Sphingosine | 0.28 | 0.75 | ||||
| Sphingomyelin | 0.28 | 0.13 | ||||
| Synthetic Cardiolipin | 0.29 | 0.20 | ||||
| Lyso-Phosphatidylglycerol | 0.54 | 0.20 | ||||
| Lyso-Phosphatidylethanolamine | 0.45 | 0.20 | ||||
| Monolysocardiolipin | 0.45 | 0.34 | ||||
| Lyso-Phosphatidic Acid | 0.40 | 0.01 | ||||
| Dilysocardiolipin | 0.32 | 0.21 | ||||
| Lyso-Phosphatidylinositol | 0.29 | 0.03 | ||||
| Lyso-Phosphatidylcholine | 0.22 | 0.08 | ||||
| Lyso-Phosphatidylserine | 0.18 | 0.02 | ||||
Notes:
- Rf values represent relative migration only, whereas absolute values depend on various environmental parameters (e.g. temperature, humidity) which may vary depending on location.
- *sf – solvent front
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