I have an intracellular metabolite which contains both hydrophilic and hydrophobic compounds. What is the procedure for separating hydrophilic compounds and hydrophobic compounds?
For small scale reverse phse chromatography, for large scale (or for a pre- purification step), liquid-liquid extraction. The exact protocol will depend on scale and how messy your initial preparation is.
Sanhita Saha
The simplest technology for separating hydrophobic and hydrophilic compounds is their distribution between two immiscible phases, for example, water and hexane or petroleum ether (it is cheaper). Mix your metabolites with these substances well in a separatory funnel. Let the mixture settle and separate the two layers: hydrophilic at the bottom, hydrophobic at the top. Remove solvents by distillation if metabolite substances do not decompose when heated. If they decompose, remove the solvent under vacuum. There are tables of distribution coefficients of substances between water and octyl alcohol. They will be useful to you for various comparisons.
Oscar & Yuri have done a bang up job of answering the question. A small but sometimes important detail is ionic strength, for instance a substance that partitions relatively equally between organic and aqueous phases can often be coerced to seek one phase or the other by adjusting the ionic strength. The classic is to add salt to the aqueous phase, quite a bit of salt to encourage the more non-aqueous components to actually disentangle from the aqueous phase and seek the organic phase. Other than that, Oscar & Yuri have covered it pretty exhaustively. This is often relevant when one is using buffers.
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