Question

In order to separate the two dyes bound the column a solution is needed with a substance that has higher affinity for the column than the dyes. Isopropyl alcohol is such a substance. If a solution of water with 70% isopropyl alcohol is applied all the purple dye elutes (comes off) the column and the dyes do not separate. In fact this is how students clean the column before reuse of the column. Read your protocol and write down the other two concentrations of isopropyl alcohol here. Why are we using two different concentrations of isopropyl alcohol?

In order to seperate the two dyes bound the column in a Sep Pak c18 Cartridge during purification a solution is needed with a substance that has higher affinity for the column than the dyes. Isopropyl alcohol is such substance. what other two concentrations of isopropyl can? and when can we use those two different concentrations of Isopropyl alcohol

Answer 1

Introduction

Grape soda usually looks purple, not because of grapes, but because of the two different food colorings in it, blue 1 and red 40. While you cannot tell that those two different food colorings are in it just by looking at the purple soda, you may be able to use a do it yourself version of a chemistry technique called column chromatography to separate the two food colorings and see them for yourself.

Chromatography is a group of techniques that are used to separate different chemicals in a mixture based on certain characteristics, or chemical properties, they have. For example, if a scientist wants to study a specific protein that is in a sample of human blood, they might be able to use chromatography to isolate it from everything else that is in the blood. Column chromatography is a chromatography technique that typically uses a long, vertical, transparent tube, or column. It is filled with small particles (in the form of a dry powder or a wet slurry) that are tightly packed. A mixture of chemicals is poured in the top of the column, and at the bottom, the different chemicals should come out separately. See Figure 1 for a diagram of this process. Here are details on the key components that a typical column chromatography setup has:

●Stationary phase: The stationary phase is made up of the small particles that the column is filled with. The particles remain stationary, or do not move, while liquids flow through the column. The particles are typically some kind of modified silica, also known as silicon dioxide, that interacts differently with the different chemicals in the mixture. Sand is commonly made of silica.

●The mixture to be separated: This is typically a mixture of different chemicals. For example, it could be a sample of human blood. Often, a researcher wants to isolate one of the chemicals, separating it from everything else in the mixture. The chemicals are separated based on their different chemical properties, and how they interact with the stationary phase and the mobile phase. We will talk about this more in a moment.

●Mobile phase: The mobile phase is a liquid that is also called the eluent. The purpose of the mobile phase is to separate the different chemicals in the mixture. To do this, the mobile phase is poured into the column and moves down through the column, flowing with or without the different chemicals in the mixture. It selectively moves the chemicals because the mobile phase is a solvent, or a type of liquid that dissolves specific chemicals.

●Collection containers: Underneath the column, a collection container—such as a cup or test tube—is placed to collect the chemicals as they flow out of the column. All of the liquid that comes out the bottom is called the eluate (including what was formerly the mobile phase and the mixture to be separated). Usually, several containers are used over time.

Figure 1. In a column chromatography setup, the column is first filled with a stationary phase (often similar to sand). (Sometimes the stationary phase is then equilibrated, which prepares the column for adding the mixture, by adding some of the mobile phase.) The mixture that will be separated (like grape soda or human blood) is then added at the top, and the mobile phase (or eluent) is added to wash the mixture through the column. The different chemicals should separate in the column and come out at different times through the bottom of the column, as the eluate, into a collection container.

The chemicals should become separated in the column and flow out the bottom at different times, allowing you to collect each one in a different container. Each different container of collected eluate is called a fraction. The result is several containers that each have a different, isolated chemical from the mixture. Researchers can then use the cup (or cups) that has their chemical of interest. Watch this video to see how column chromatography is commonly done in a laboratory. Note: The column chromatography method in this science project is much simpler than how it is done in this video, but many basic principles of how it is done are the same.

Figure 2 shows a diagram of the general column chromatography process, as was described in detail in the video. In Figure 2, you can see that after the stationary phase (gray) is added, the mobile phase (light blue) is added to equilibrate the column. Next, the mixture to be separated (purple) is added, followed by additional mobile phase to wash the mixture through the column. The components of the mixture (blue and red) are separated in the column and end up in different collection cups underneath the column.

Ideally, in column chromatography, the different chemicals in the mixture become separated in the column and come out the bottom at different times. For example, in this diagram, the red chemical travels faster through the column and comes out before, and separately from, the blue chemical (as they are washed through the column with the light blue mobile phase). Different containers are used to collect the eluate so that the chemicals are separated.

So what makes the chemicals separate in the column? In other words, why do some chemicals flow faster through the column than others? As previously mentioned, the chemicals can be separated by their chemical properties. One chemical property that is often used for this is chemical polarity, which refers to how the negative and positive charges are separated in a chemical. For example, water is a polar chemical because it has slightly different charges on different places; its oxygen has a slight negative charge and its two hydrogens have a slight positive charge, as shown in Figure 3. On the other hand, a chemical can be nonpolar if its charges are spread equally throughout the chemical. Oil and ethane are both good examples of nonpolar chemicals.

In this science project, you will make your own homemade column chromatography setup and determine whether your setup can separate the blue and red food colorings from a sample of grape soda. To do this, you will be making a complex, powerful piece of laboratory equipment at home, using relatively inexpensive parts!

You will use a nonpolar stationary phase, which should interact with, and grab onto, other chemicals that are nonpolar, like blue 1 and red 40. But how well it grabs them depends on how nonpolar they are, so it should hold onto the blue 1 slightly better than the red 40. You will use a specific product called Space Sand as your stationary phase. This will be packed into a plastic syringe, serving as your column. Space SandTM (also known as Magic Sand) is silica that has been modified with a nonpolar coating, making it hydrophobic (specifically, it has had chains of carbon and hydrogen added to it). To see just how hydrophobic Space Sand is, see Figure 4 which shows a picture of a water droplet on some of this sand. It is commercially sold as a toy because it is fun and it is fascinating to watch how it interacts with water. Space Sand is similar to nonpolar stationary phases used in laboratories—both are silica that has been modified to be hydrophobic—but Space Sand is not quite as hydrophobic as the modified silica usually used in laboratories. Will the Space Sand be hydrophobic enough to separate the blue 1 and red 40? For your mobile phase, you will use 70% isopropyl alcohol, which is more nonpolar than pure water and can act as a nonpolar solvent, so it should dissolve nonpolar chemicals that are in the stationary phase, letting them flow through the column.