I instructed an undergraduate to make a solvent system (1:1 Chloroform:n-Hexane). This solvent system with a hexadecane internal standard was used to make extracts from ground walnut bark (fresh, containing water). The extract was made by using 1mL solvent system with 200mg ground bark, with 2 pulls. The supernatants were combined and filtered.
All of the extracts formed 2 layers and I have no idea why. Chloroform and hexane should be miscible with each other and immiscible in water. Based on this, one would expect 2 layers, but the water layer should be extremely small. However, it looks like the two layers are about equal volumes.
I added 1mL of water to one of the extracts in a larger vial to see if the water would become miscible with one of the layers. After vigorous mixing, it does seem like two layers were made after settling, and the water layer appears to be thicker.
Was there a mistake made with the solvent system? I am not sure what is going on.
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