Column Chromatography: Can you Separate the Dyes in Grape Soda Using Space Sand™?
AbstractWhat color is grape soda? If you pour it into a clear glass you can easily see it is purple, but that is usually not its natural color. Manufacturers add red and blue dye to the soda. The dyes mix together and you get purple soda. What if you wanted to un-mix the dyes, could you? Yes! In a chemistry laboratory, using a technique called column chromatography, you could separate the two dyes again. But what about at home, can you use low-tech supplies to do the same thing? In this science project you will try to do just that using some interesting materials including a toy/craft product called Space Sand.
Teisha Rowland, PhD, Science Buddies
Thanks to Andrew Bonham, PhD, Metropolitan State University of Denver, and volunteers at Thermo Fisher Scientific and the Science Education Council at PPG Industries, for feedback and advice on this science project.
- Space Sand™ is a registered trademark of DuneCraft, Inc.
Investigate whether a homemade column chromatography setup can be used to separate and isolate the different food colorings that are in grape soda.
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.
A tube with a hole at the bottom is filled with a tightly packed mixture of solid particles called a stationary phase. A solvent liquid called eluent is added to the top of the container and slowly seeps through the solid material, moving downward towards the bottom of the container. When the liquid reaches the bottom of the container it drains through a small hole and the liquid that exits is called the eluate.
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.
Figure 2. 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.
Figure 3. Water (H2O) is a polar chemical because its negative and positive charges are separated. Ethane (C2H6) is a nonpolar chemical because its charges are spread out equally.
A chemical's polarity can affect its solubility, or how well it dissolves in different solvents. Generally, nonpolar molecules are hydrophobic, meaning they do not dissolve well in water (think of how water and oil do not mix), while polar molecules are hydrophilic, meaning they do dissolve well in water. Overall, this means that a given mobile phase may dissolve a certain chemical and make it flow quickly through a column, along with the mobile phase itself, but that same mobile phase may not dissolve another chemical, leaving it stuck (or moving slower) in the column. This is how chemical polarity can be used to separate two different chemicals in a mixture.
Let us get back to the question of whether you can use column chromatography to separate some common mixtures, such as artificially colored beverages like grape soda. As mentioned, grape soda contains two different food colorings, blue 1 and red 40. (Typical grape soda contains many other ingredients, too, but these are harder to see; if you could separate the dyes, it would be much easier to see if your efforts were successful.) Food coloring, or food dye, is made up of dye molecules. (You can confirm that a food has these dyes by looking at the ingredients lists on the packaging, often listed as "FD&C" followed by a color and number, such as "FD&C Blue No. 1," or sometimes just listed as "blue 1.") Blue 1 and red 40 are both relatively nonpolar. However, red is slightly more polar than blue. This slight difference is enough for a chromatography column in a research laboratory to separate the different dyes, but could you do this with a homemade setup?
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.
Figure 4. A water droplet on Space Sand balls up because the sand is very hydrophobic.
Pop open a can of grape soda and get ready to find out whether your homemade column chromatography setup can separate the food colorings inside!
Terms and Concepts
- Column chromatography
- Stationary phase
- Silica, or silicon dioxide
- Mobile phase, or eluent
- Column chromatography fractions
- Chemical polarity
- Food coloring
- How can column chromatography be used to separate and isolate different chemicals in a mixture?
- What is an example of something that is hydrophilic? What about something that is hydrophobic?
- In this science project, which dye do you think will come out of the column first, blue 1 or red 40? Why?
- What is Space Sand? How is it different from normal sand?
- Is isopropyl alcohol more or less polar than water?
You can do further research by visiting the following websites, which give information about hydrophobic sand, column chromatography, and polarity of chemicals:
- Wikipedia Contributors. (2013, July 26). Magic sand. Wikipedia: The Free Encyclopedia. Retrieved August 7, 2013.
- University of Colorado at Boulder. (2013, May 13). Column Chromatography. Department of Chemistry and Biochemistry. Retrieved August 7, 2013.
- School for Champions. (2013, May 27). Polar and Non-Polar Molecules. Retrieved August 8, 2013.
- Read, D. (2008, September 16). Column chromatography. Retrieved July 30, 2013.
This science project was inspired by the following lab experiment:
- University of Minnesota. (2002, June 10). Separation of Food Dyes Via Column Chromatography. Retrieved July 2, 2013.
Materials and Equipment
- Plastic syringes with Luer-Lock, 30 mL, without needles (4); available online at Amazon.com
- Space Sand (24 mL, or about 2 Tbsp.); available at some toy stores or online at Amazon.com. White sand is recommended so that dyes from the sand do not potentially interfere with results.
- Cotton rounds (2)
- Optional: Funnel with a tip that fits into the top of the syringe once the plunger is pulled out
- Clear plastic cups or drinking glasses, at least 5-oz. (21)
- Permanent marker or sticky note and pen or pencil
- Safety goggles
- Isopropyl alcohol (150 mL)
- Distilled water (105 mL); available at most grocery stores
- Grape soda (30 mL)
- Lab notebook
- Optional: Digital camera to take pictures of your experiment for your project display board
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Using Column Chromatography on Grape Soda
- In your lab notebook, make data tables like Table 1 and Table 2. You will be recording your observations in these data tables.
- Remember that the eluate is the liquid that comes out the bottom of the column, and that each different cup in which the eluate is collected is referred to as a different fraction.
|Colors of Eluates Observed During the Experiment|
|1st Fraction||2nd Fraction||3rd Fraction||4th Fraction||5th Fraction|
|Colors of Column Sand Observed During the Experiment|
|Sand After Equilibration||Sand After Collection of the 1st Fraction||Sand After Collection of the 5th Fraction|
- Make sure to wear safety goggles throughout the procedure to not get any back splash of isopropyl alcohol into your eyes.
- Get out three plastic cups or drinking glasses. Pour some 70% isopropyl alcohol into one, distilled water into another, and grape soda into the last cup.
- Specifically, you will need at least 150 mL of the isopropyl alcohol, 105 mL distilled water, and 30 mL grape soda. But you do not need to carefully measure out these quantities now because you will measure them later, as you use them.
- You could label the cups (with either a permanent marker if they are plastic or with a sticky note and pen or pencil if they are glass) or just keep them near the containers they came from so you know what is in each cup.
- Get out six more plastic cups and label them Waste, 1st Fraction, 2nd Fraction, 3rd Fraction, 4th Fraction, and 5th Fraction. It will be easiest to arrange them in a line in front of you, going left to right.
- Remove one of the 30 milliliter (mL) syringes from its packaging. Use a permanent marker to label the syringe "A" on the side somewhere.
- Cut a small circle out of a cotton round so that it just fits into the syringe. Pull the syringe's plunger all the way out and insert the cut cotton round. You can use a pen to push the cotton pad all the way down into the tip of the syringe
- Fill the syringe with 8 mL of packed Space Sand, as shown in Figure 5. The cotton pad will prevent sand from falling out of the syringe.
- Tip: To fill the syringe with the sand, it might be easiest to use scissors to cut off a small corner of the sand's bag and slowly pour it out from that corner, and into the syringe. Alternatively you may use a funnel.
- As you fill the syringe with the sand, repeatedly tap the syringe on a hard surface so that the sand becomes compacted. If needed, use the syringe plunger to push sand down and off the inner sides of the syringe.
- When you think you are done, make sure that the sand is not less than 8 mL after you have tapped the column several times.
- Keep in mind that it is much better to have a little too much sand (such as 8–9 mL) in the syringe than too little sand (less than 8 mL). Why do you think this is?
- When it is ready, carefully set the sand-packed syringe aside for now.
Figure 5. Fill one of the syringes with 8 mL of Space Sand, tapping the syringe as you fill it so that the sand is compact.
- First equilibrate your column with the mobile phase and some distilled water. This will prepare it for processing the grape soda.
- If it is not already, pull the plunger out of the sand-packed syringe, which you labeled "A," and carefully set the syringe in the cup labeled Waste.
- Get out a second 30 mL syringe. Use a permanent marker to label this syringe "B." Use syringe B to suck up 30 mL of 70% isopropyl alcohol from its cup.
- Hold syringe A (the sand-packed syringe) over the waste cup. Then, in your other hand, hold syringe B over syringe A and slowly push out the alcohol from syringe B into syringe A. You might need to pour the liquid into the column in increments so it does not overflow. See Figure 6 to see how the syringes should be arranged. Note: Be careful to not disturb the sand too much while adding the alcohol.
- Let the alcohol drip out of syringe A and into the waste cup below.
- To speed up the process, you can put syringe A's plunger back into the syringe and slowly push out the remaining alcohol once you have squeezed it all out of syringe B.
- Note: When doing real column chromatography, it is often important to never let the column run dry. In other words, once a column is damp, it should stay damp. However, in this science project it is okay to push the liquid out of the sand if the sand will be dry for just a minute or two. While, in general, it is better to let the sand stay a little damp, there should be no remaining liquid above the level of the sand in the syringe.
Figure 6. Hold syringe A (the sand-packed syringe) so that it is over the waste cup. Then hold syringe B (the alcohol-filled syringe) so that it is above syringe A, and slowly push down on syringe B's plunger so that the alcohol goes into syringe A. You will probably need to use two hands to do this.
- Once all the alcohol has dripped out, slowly remove the plunger out of syringe A, but continue to hold it over the waste cup. Tilt the plunger back and forth so as it does not "pop" when you pull it out.
- Take syringe B and use it to suck up 30 mL of distilled water.
- Repeat step c. with the distilled-water-filled syringe (instead of the alcohol-filled syringe).
- Your column has now been equilibrated and it is ready to use with the grape soda. Record the color of the equilibrated sand in the data table (the one similar to Table 2) in your lab notebook.
- Now add a sample of grape soda to your equilibrated column.
- Carefully pull the plunger out of syringe A and set the syringe in the cup labeled 1st Fraction.
- Take syringe B and use it to slowly suck up 10 mL of grape soda. Note: Be sure you slowly suck it up to prevent bubbles from accumulating in the syringe.
- Hold syringe A over the 1st Fraction cup. Then, in your other hand, hold syringe B over syringe A and slowly push the grape soda out of syringe B and into syringe A, as you did to equilibrate the column. Try not to disturb the sand too much.
- You can use syringe A's plunger to speed up this process, but remember not to leave the sand dry for more than a minute or two.
- What is the color of the eluate that comes out in this fraction? What is the color of the sand in the syringe? Write your observations in the data tables in your lab notebook.
- Next, add 5 mL of distilled water to the column for better color separation.
- Slowly remove the plunger out of syringe A by tilting it back and forth and carefully set the syringe in the cup labeled 2nd Fraction.
- Take syringe B and use it to suck up 5 mL of the distilled water.
- Hold syringe A over the 2nd Fraction cup. Then, in your other hand, hold syringe B over syringe A and slowly push out the water into syringe A, as you did before. Again, make sure to not disturb the sand too much.
- What is the color of the eluate in this step? Did it change? Note down your observations in the data tables in your lab notebook.
- Next, add your 70% isopropyl alcohol mobile phase to elute the colors from the column.
- Take syringe B and use it to suck up 10 mL of the 70% isopropyl alcohol.
- Move syringe A over to the 3rd Fraction cup. Then, in your other hand, hold syringe B over syringe A and slowly push out the alcohol into syringe A, as you did before without disturbing the sand.
- Important: Be sure you closely watch the liquid drip out of syringe A, and as soon as the drops look like they are changing color, move syringe A so that it is over the 4th Fraction cup. Then let the remaining liquid drip, or be pushed out, into this cup.
- What is the color of the eluate that you get in the 3rd and 4th Fraction cups? Record all of your observations in the data tables in your lab notebook.
- Lastly, add some more of your 70% isopropyl alcohol to the column.
- Slowly pull the plunger out of syringe A as you did before and carefully set the syringe in the cup labeled 5th Fraction.
- Take syringe B and use it to suck up 10 mL more of 70% isopropyl alcohol.
- Hold syringe A over the 5th Fraction cup. Then, in your other hand, hold syringe B over syringe A and slowly push out the alcohol into syringe A, as you did before. Make sure to not disturb the sand too much.
- If you use syringe A's plunger to speed up this process, be sure to leave the sand damp, since you do not want it to be dry while you take time to prepare for the next trial.
- What is the color of the eluate that comes out in the 5th Fraction cup? What is the color of the column's sand? Write your observations in the data tables in your lab notebook.
- Do not discard your results from each trial; you will use them for comparison when you analyze your results.
- If you want to, you can take pictures of your eluates in the different cups. You should include a sample of the original grape soda in a cup for comparison. You may want to take pictures using a white background, such as a sheet of blank paper, and include labels. You could include these pictures on your Science Fair Project Display Board.
- Repeat steps 4–11 two more times. Be sure to record your observations in the data tables in your lab notebooks, in the rows for trials 2 and 3.
- For syringe "A," use a new syringe, but you can use the same syringe as before for syringe "B."
- Use fresh cups for the waste and each eluate fraction.
- Repeat step 3 if you need more liquid in those cups.
Analyzing Your Results
- When you have completed the column chromatography, compare the liquids in the cups and the results you recorded in the data tables in your lab notebook.
- Are the colors of the eluates different? For example, are the fractions collected from when you added the grape soda different from the fractions collected when you added the distilled water or the 70% isopropyl alcohol? If so, how are they different? Why do you think this is?
- Were your results consistent between your different trials, or was there some variability?
- If the eluates from the different fractions are different colors, what do you think this has to do with how hydrophobic the different food colorings in the grape soda are? Hint: Try re-reading the Introduction to figure this out, thinking about how 70% isopropyl alcohol might interact with the different food colorings in the grape soda.
- Overall, was your homemade column chromatography setup successful at separating the food colorings in the grape soda? If it worked, can you explain how?
Ask an Expert
- Many different artificial beverages, dry beverage mixes, and other foods contain different food colorings. Use your column chromatography setup to separate food coloring mixtures in other beverages or food. Do you need to change the stationary and/or mobile phase(s) for it to work?
- In this science project, you created a homemade column chromatography setup that may have worked well, but can you make it work even better? For example, could changing the amount of grape soda, type of mobile phase, number of collection cups used, or amount of sand in the column improve your results? (Important: If you use a different mobile phase, be sure to take the proper safety precautions for using any of the chemicals). What about trying different concentrations of your mobile phase? You could check out the resources in the Bibliography in the Background for more information on column chromatography and to get ideas of how to improve your setup.
- Make a column chromatography setup that is even easier for people to make at home. For example, you probably had to order syringes to do this science project. Could you replace them with something that is more common, such as plastic water bottles that have their bottoms cut off and are flipped upside down? If you do this, how can you prevent the sand from leaking out? Since the bottle is probably larger than the syringe, will you need to increase the amount of materials (e.g., sand, grape soda, isopropyl alcohol, etc.) that you use? Can you let the mobile phase flow out just by using gravity? There can be a lot of challenges, but making a technique like this available to more people can be very rewarding!
- You can make your own hydrophobic sand at home and then compare how it works in your homemade column chromatography setup to how the Space Sand worked. To make hydrophobic sand, make a thin one- to two-grain-thick layer of sand on a cookie sheet and spray the sand with a spray-on water repellent. Be sure to work in a well-ventilated area, follow all safety precautions, and use a cookie sheet that can be discarded afterwards (you should not use it with food). You will want to repeat this process several times to thoroughly coat the sand: Mix the sand after spraying it to spread around the spray, let the sand dry, turn the sand over and spray it again. Let the sand completely dry before testing it with a drop of water to see if it is hydrophobic (if it is, it should look similar to Figure 4 in the Introduction). Does it work as well, or better than, the Space Sand?
- For other science projects on chromatography see:
- For other science projects on food coloring dyes see:
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