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Name: Unknown
Status: student
Grade: 9-12
Location: DC
Country: N/A
Date: August 2006

Question:
Why is a mixture of acetone and water used to separate the non water-soluble materials in a chromatography lab?



Replies:
Hi, In general, acetone is a very useful solvent and will dissolve a great number of organic molecules (exceptions are very greasy molecules). Since the acetone is also miscible with water, as the percentage of acetone in the acetone water mixture increases, it increases the solubility of non-water-soluble molecules in this new solution. Solubility can be measured in mg/mL and a correlation can be made between the solubility of a compound in water, in acetone and in different water/acetone mixtures. If a compound is completely insoluble in water ( < 0.1mg/mL) and decently soluble in acetone (5-20 mg/mL), then as you add water to the acetone, eventually the compound should crash out of solution as a precipitate (solid or gum) due to the lack of solubility in water. The higher the solubility in acetone, the more water you will have to add to crash the solid out--if it can even be crashed out. Remember that as you add water, you are also increasing the volume and hence diluting the mixture, which helps solubilize the solid. This is one way that you can separate things, but high purities are sometimes difficult to obtain. This is the primary use for a chromatography lab--to separate those mixtures that we cannot separate using more basic, scalable, quicker and cheaper methods (solubility, extractions, acid/base chemistry/pH control, distillations, etc.)

If a chemist is using an aqueous based solvent system (i.e. water and a water-miscible organic solvent--usually limited to acetonitrile, methanol, tetrahydrofuran or acetone), then he is using a "reverse-phase" system. A normal phase system (only termed normal phase because it was invented first) uses two organic solvents, usually one very non-polar like hexane or heptane, and one that is more polar like ether, dichloromethane, ethyl acetone or methanol. The system that is used depends on the relative polarity of the molecules that he is trying to separate.

Having stated that, to succinctly answer your question, a chemist would use a reverse-phase system because it offers different selectivity than normal-phase systems. Reverse-phase systems are generally used on HPLC instruments (high pressure liquid chromatography), where the columns that they use are very small (5 to 15 cm long) and have a very high number of theoretical plates over that distance. There are a lot of different types of reverse-phase columns that one can use to screen separation conditions. Once an optimal separation is discovered, then the chemist can do "prep-HPLC" (preparative-scale, which means that instead of injecting micrograms of material, he can inject several grams to separate and isolate larger quantities of the different compounds in the mixture so that further tests can be run to identify and characterize these, often times, low concentration and unknown compounds. In this manner, a chemist can use chromatography to see what byproducts are being formed in a reaction and start to theorize a mechanism for the reaction pathway that incorporates the identified impurities being formed.

Matt Voss



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