Molecule of the Day

The previous post about DEAE cellulose reminded me of a classic experiment in chromatography: the separation of dyes. This is where the name comes from - even though most of what we separate anymore isn’t colored, it stuck. Mikhail Tsvet, a Russian chemist, used it to separate chlorophylls and carotenes, two classes of brilliantly colored biomolecules. Lucky for you, when I have nothing to do on a Sunday afternoon, I do…well, the exact same thing I do during the week, only for free. In my kitchen. It gets messy. Today wasn’t so bad.

I went to the grocery and bought the regular food coloring everyone gets, the four little plastic bottles with red, blue, green, and yellow. If you look at the ingredients, my box says there is: water, propylene glycol, yellow 5, red 40, and blue 1. Propylene glycol, mentioned on the fomepizole entry, is just a nontoxic alcohol that probably makes the dyes a bit more soluble. Notice there are only three dyes. This is probably not so surprising since we have yellow, red, and blue, and the only one without its own dye is green. Presumably the venerable principle of “yellow and blue make green” is at work here. We set out to verify the YABMG theory.

Here is the idea behind chromatography: you have a stationary phase and a mobile phase. Stationary phases are, for example, DEAE cellulose and silica, as mentioned the other day. The idea here is that whatever molecule you want to use will stick to the stationary phase to some degree. Today, we use regular old 20 pound cheap printer paper as our stationary phase.

A little explanation. You are looking down into a regular drinking glass. The paper has little spots of food coloring on it, as small as I could make them. Notice I didn’t do so hot with old bluey. In the bottom is 70% isopropyl alcohol/30% water, from the drugstore.

This alcohol/water mixture is the “mobile” phase. The idea here is that there is a solvent that competes a bit to move the molecules along the paper. Things that interact more strongly with the solvent than the paper move quickly; things that interact more strongl with the paper than the solvent move slowly. We rest the paper in the alcohol, with the alcohol touching the paper but not the spots of dye. As the solvent wicks up the paper, the dyes begin to move:

Notice that, as we expected, the green is separating into yellow and blue. Surprisingly, the yellow seems to have a tint of red! Presumably it just makes the uncut yellow 5 a little less Mountain Dew-yellow and unappetizing, and if you’re after that color, you’ll have to buy your food coloring at the “Extreme Everybody to the Limit!!!” grocery. Also note the use of aluminum foil at the top of the glass. This keeps the mobile phase (alcohol/water mix) from evaporating and makes the chromatograph develop much faster.

Here, I have removed the paper from its glass and let it dry. You can see where the solvent made it before I removed it; that line up top (it’s purpley on the right).

As we saw at the very beginning, the “yellow” was in fact yellow and a bit of red. The pink spot seems to have made it the same distance as the other red spot. We know there is only one red dye based on the ingredients list, but if we didn’t, this would be evidence in support of them being the same.

The green lane has separated into yellow and blue; both yellow spots migrated the same distance, as did both blue spots. On the right is a paper with only green applied; the resolution is a bit cleaner between the two spots.

The red lane has the same migration distance as the pink spot in the yellow lane and has only one component.

You’ll notice that my fat-finger-applied blue spot has become an ugly blob. The appearance of two shades is probably a red herring; there was simply too much dye on the paper to move cleanly, and it was in too big a spot. It may actually really be red dye, though. Many people use coffee filters, paper towels, and the like for this experiment. I could not get clean spots with these and only the relatively low-absorbance copy/printer paper gave me satisfactory results. You want the smallest spot you can get, because if two spots overlap, you won’t be able to tell if they’re different!
Finally, notice that different dyes migrate different distances. The blue follows the solvent front almost exactly, the red is slightly retarded, and the yellow sticks pretty strongly to the paper and barely moves at all. Also note that there is a little purple line at the solvent front (visible only in the right paper). This is related to the fact that the parts of the paper that have encountered the alcohol/water mixture look less white. There are actually dyes in even “bright white” paper!

The next few days will entail a further discussion of how this all works. Try it at home! See you tomorrow.