MadSci Network: Biochemistry
Query:

Re: Why does adenine have more mobility in a chromatograph then cytosine?

Date: Fri Oct 23 11:36:47 1998
Posted By: Michael Onken, MadSci Admin
Area of science: Biochemistry
ID: 908766819.Bc
Message:

Chromatography has many forms and uses: Gas Chromatography, High Pressure Liquid Chomatography (HPLC), Thin Layer Chromatography (TLC), gel electrophoresis (several forms), and dozens of column chromatographies, including affinity columns, ion-exchange columns, gradient columns, and gel- phase columns; all of which I've had to use for the various assays and isolations that are the foundations of molecular biology and biochemistry. The majority of these techniques do not separate molecules based on size. Unfortunately, the most commonly used form of chromatography, gel electrophoresis, does separate based on size, or more specifically mass, so many people see these as the paradigm for chromatographic separation. In fact, the more traditional gel-bead columns size-separate in the opposite direction, i.e. larger molecules migrate faster through the beads than smaller molecules! So, there was no experimental error in your separation: adenine was supposed to migrate faster than cytosine.

But why? Well, here are some data on the two molecules, that I lifted from the Merck Index:

Adenine:
MW = 135.14
1 g dissolves in 42 ml water at neutral pH
slightly soluble alc.
insoluble ether, chloroform

Cytosine:
MW = 111.10
1 g dissolves in 130 ml water at neutral pH
slightly soluble alc.
insoluble ether

If you'll notice, adenine has just over a fifth (21%) more mass than cytosine, however it is over three times (3.1) more soluble in water than cytosine. So separating the two based on their solubility would be much easier than separating them based on their mass. Since they are each only slightly soluble in alcohol, a solution of mostly alcohol and a little water (say 4:1 alcohol:water) would further amplify their solubilities, especially against a solid substrate. That's how TLC works: you spot your sample onto one end of a plate covered with silica and immerse that end in the solution; as the solution moves across the plate, it carries the molecules with it, however the low solubility causes the molecules to constantly fall out of solution back onto the silica; so whichever molecule can stay in solution the longest (i.e. is more soluble) will travel the farthest along the plate. Since adenine is more soluble than cytosine, you would expect it to have a higher Rf under these conditions.


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