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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

 

Solvent Systems
Ratio(V:V)
i
Chloroform:Methanol:Water
65:25:4
ii
Chloroform:Methanol:Ammonium Hydroxide
65:25:4
iii
Chloroform:Hexane:Methanol:Acetic Acid
50:30:10:5
iv
Toluene:Pyridine:Water
60:60:10
v
Cyclohexane:Ethyl Acetate
3:2
vi
Toluene:Chloroform:Methanol
85:15:5

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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