What is Capillary Electrophoresis?

Instructor: Darla Reed

Darla has taught undergraduate Enzyme Kinetics and has a doctorate in Basic Medical Science

In this lesson, you will discover what electrophoresis means and how capillary electrophoresis works. You will also learn how molecules flow and how data is plotted.

What is Capillary Electrophoresis?

Let's begin this lesson by the two words that make up the term 'capillary electrophoresis.'

Have you ever gone through a tunnel? A capillary is a very small tube structure similar to a tunnel.

Capillary

The word that follows, electrophoresis (elect-roe-for-ee-sis), is not actually as complicated a concept as it may be to pronounce - it works much like a magnet. There are two sides to a magnet: a north and south pole. Different poles attract while the same poles repel each other. In electrophoresis, there are two sides, just like a magnet. One side has a positive charge (+), the anode. The other has a negative charge (-), the cathode.

Electrophoresis has two ends.

Molecules come in many sizes and can have a positive, negative or neutral (no charge) state. When molecules have a charge, they are called ions.

Molecules have charge and size

Since molecules can have different charges, in electrophoresis, molecules can be pulled toward the anode or cathode using electricity. If filters are put in the path of molecules being pulled, then they can be separated not only by charge, but also by size. Therefore, electrophoresis is a technique used to separate molecules based on size and charge.

Electrophoresis separates by charge and size.
Electrophoresis separates by charge and size

It is often used to separate proteins, RNA and DNA, but electrophoresis can also distinguish other types of molecules.

Capillary electrophoresis (CE), then is the separation of molecules using electricity and a very small tube called a capillary.

How Does Capillary Electrophoresis Work?

The capillary, which as we discussed is shaped like a tube, is open on each end, and each end is placed in a buffer. Have you ever tried to ride a water slide without the water flowing through it? It would be difficult and certainly less fun. A buffer is like the water in a water slide - it is simply a vehicle used to carry molecules.

Two ends of the capillary are in buffer solution.
CE setup- two end of capillary are in buffer

Capillary tunnel walls have a negative charge. The buffer usually has a positive charge. Since the walls of the capillary are negative, the buffer is attracted to the tunnel walls.

Positive buffer neutralizes negative capillary walls.
Capillary negative wall is neutralized by positive buffer

This is what lets molecules flow evenly through the middle of the tunnel, just like the water in a water slide lets you flow easily down without worrying about your skin creating friction against the walls.

Also in the buffer, at each end of the tunnel, are electrodes (the anode and cathode). Electrodes are like the roads leading into and out of a tunnel. The electrodes connect to a power source - when the power source is turned on, traffic through the tunnel will move a certain way.

When studying CE, scientists typically create conditions in which molecules move from the anode to the cathode. When the power is on, molecules tend to go with the flow of traffic. Since the buffer is often positive, traffic will flow in the direction of the cathode.

Molecules flow in the capillary when power is on.
Electrodes are placed in the buffer and buffer flows when power is turned on.

Molecule Flow

Imagine you're running a race through a tunnel. At the finish line, there is free ice cream. People who love ice cream will race through the tunnel faster than those who don't like it. People who are in it just for the love of running and do not like ice cream will likely be a bit slower.

Ions migrate through the capillary tunnel in a similar way. When the power is turned on, the positive ions will run to the cathode just like the ice cream lovers will race to the finish line. Neutral ions, behaving like those who enjoy running but not ice cream, will come up behind the positive ions. Negative ions are like the runners who don't like ice cream and don't enjoy running.

The order ions arrive at the cathode, then, depends on charge. Size also plays a factor in ion flow - the smaller the ion, the faster it reaches the cathode, as depicted by the following image:

Molecule flow in CE depends on ion charge and size
Molecule flow in CE

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