Molecule of the Day

You have probably heard of chromatography. This is a chemical technique for separating mixtures of compounds. Various “stationary phases” can be used to separate compounds based on different characteristics. Probably the most common is silica, which is just pure, clean sand (speaking a little loosely. It’s so pure you wouldn’t recognize it as sand. It’s a fine white powder and very homogeneous in size.) It separates compounds based on their polarity. It is the most common medium used by organic chemists because it’s relatively cheap and it works on a wide variety of substrates.

Biochemistry makes it trickier. First of all, everything’s dissolved in water, which is the most polar solvent most people will ever encounter. Silica chromatography with water just doesn’t work. It’s done with organic solvents like ethyl acetate and methylene chloride. Biomolecules, as a rule, don’t take well to being dissolved in anything but water. A lot of the time (especially with proteins), you’re worried about a specific three-dimensional structure. Organic molecules, as a rule, don’t really care. This is largely because most of them are too small to have enough freedom to fold into anything interesting. This is why I can show the daily molecule as a stick drawing and not have to worry about 3D structure.

Because of this, biochemists are forced to use different stationary phases/resins instead of silica. One common one is DEAE cellulose (diethylaminoethyl cellulose):

As you can imagine, it’s made from cellulose, largely because the stuff is so cheap. The business end is that diethylaminoethyl moiety. In water, at any pH below the pKa of 11.5 (just about any pH you would ever use; biologists usually work between pH ~5 and ~8, 7 being “neutral”, less being acidic, more being basic), it is predominantly in that form on the right. It is positively charged and pretty good at binding anions (negatively charged species) to its surface. You can take hundreds of milliliters of solution containing less than a milligram of your favorite anion, and flow it over a few mL of the resin (it is present as sort of a slurry - almost exactly like oobleck.) Almost all your anion will stick to the resin. It can then be eluted (washed off) with a buffer with a lot of salt.

Let’s say we’re using sodium chloride and we’re purifying DNA. Initially, when we load the column with the DNA, it should be dissolved in a low-salt buffer (preferably a buffer like tris rather than phosphate, since phosphate is itself an anion and will stick to the column, competing with our DNA). We can wash the column repeatedly with low-salt buffer (say, 100mM) and very little DNA will come off, but that which doesn’t stick will. If we start washing high-salt buffer over it (say, 2M), the chloride ions will start competing with the DNA for binding those positively charged diethylammoniumethyl moieties, and the DNA will wash off the column.

The principle works similarly for proteins, but you will undoubtedly use different concentrations of salt - DEAE works especially well for DNA because DNA has so much more negative charge than anything else. That 100mM salt would probably elute many proteins, which is great; we’re left with only DNA.

That one might have been a little more in depth than usual, sorry if I lost you. Night.