Experimenting with various separation techniques (e.g. electrophoresis)

Introduction: (Initial Observation)

Most organic material found in the nature, extracted from plants or from other live organisms are not pure. Instead they are a mixture of two or more organic substances. In order to identify each organic substance we first need to separate them and get pure samples of each substance. We can then use other techniques to identify each substance based on it’s chemical and physical properties.

Depending on the type of chemicals, different methods may be used for separation. Among these methods are: Chromatography, distillation, electrophoresis, crystallization, filtration and others. Electrophoresis is the method used to separate chemicals using electricity.
In this project you will attempt to separate chemicals using your own electrophoresis equipment and try to troubleshoot and improve your electrophoresis skills.

Dear

This project guide contains information that you need in order to start your project. If you have any questions or need more support about this project, click on the “Ask Question” button on the top of this page to send me a message.

If you are new in doing science project, click on “How to Start” in the main page. There you will find helpful links that describe different types of science projects, scientific method, variables, hypothesis, graph, abstract and all other general basics that you need to know.

Project advisor

Try to improve the procedures suggested in this page. You can modify any part to see how does it affect your results. Changing voltage, Changing electrodes from copper to titanium or aluminum, changing the electrolyte are among the changes that you can try.

Information Gathering:

Find out about different methods of separating chemicals. Read books, magazines or ask professionals who might know in order to learn about electrophoresis and factors affecting the performance and the results of different electrophoresis methods. Keep track of where you got your information from.
Electrophoresis is a separation technique. It is based on the different mobility of ions (molecules which are charged) in a support which is subjected to an electric field. Ions run more or less quickly along the substrate according their charge, size, shape, etc.

Commonly used methods of electrophoresis are Gel electrophoresis, capillary electrophoresis and paper electrophoresis.

According to the technique which is used, the apparatus consists of two small basins which contain an electrolyte, a support (i.e.: filter paper, cellulose acetate strips, polyacrylamide gel, or a capillary tube), an electrical DC power supply and two electrodes. Electrophoresis is widely used to separate substances such as amino acids, proteins, strands of DNA, etc. As in the case of chromatography, people use different supports and solvents according to the substances to be separated and the techniques used.

PAPER ELECTROPHORESIS

C. L. Stong, on the magazine “Scientific American” proposed an experiment on paper electrophoresis.

1 – As shown by the above figure, inspired by this article, place two little basins a few cm (one inch) apart. Pour in the basins an electrolyte made of a teaspoon of table salt and another of baking soda in 300 ml(1 1/2 cups) of tap water. Place a glass plate on the two basins and on it place a stripe of filter paper soaked with the electrolyte. This stripe has to be immersed with each end in the electrolyte in the basin for to complete the electrical circuit. With a pencil, draw a line across the filter paper and place a little drop of blood on it. Cover the paper with a second glass plate. Put an electrode into the electrolyte of each basin and apply 45V in direct current (from 4 to 8 Volt per cm). You can obtain this from 5, 9-volt batteries or 8, 6-volt batteries connected in series. With the passing of time, you should see 5 little spots move toward the negative electrode. These spots are made of different protein components of the plasma: globulins (alpha, beta, gamma), albumin and fibrinogen. In reality, to make these substances better visible it is necessary use a stain such as bromphenol blue. Lacking it, try red cabbage juice.

2 – Try also to separate the components of some other substances and observe what happens. Keep in mind that some of these substances may not be ionic.
LOOK OUT!: Do not use high voltages for this experiment. Never touch the electrodes, the electrolytes, or the paper stripe. Take care never to short out the two electrodes. Finally, we recommend to insert a fuse on one of the two cables. The fuse will break the circuit when there is too high a current. We suggest about 10 mA. At the end of the migration of the spots, remove the electrodes from the basins and take off the cables from the batteries.

Gel electrophoresis is a key technique in molecular biology. It uses an electric field to move charged molecules through a gel. Researchers use electrophoresis to detect a compound, to separate one compound from a mixture, to characterize a mixture, and to purify a compound.

The compounds most commonly used in electrophoresis are proteins and nucleic acids.

The most common gelling agents are agarose for nucleic acids and polyacrylamide for nucleic acids and proteins.

Question/ Purpose:

What do you want to find out? Write a statement that describes what you want to do. Use your observations and questions to write the statement.

Experiment electrophoresis and develop troubleshooting skills by planning, assembling and using electrophoresis equipment.
Grasp the importance including appropriate controls when running an assay.
Demonstrate the separation of components of a mixture;
Demonstrate the use of “reconstruction” experiments;

Here’s a question: is green food coloring dye really green?

What else could it be?

Could it be a mixture of blue and yellow?

Identify Variables:

When you think you know what variables may be involved, think about ways to change one at a time. If you change more than one at a time, you will not know what variable is causing your observation. Sometimes variables are linked and work together to cause something. At first, try to choose variables that you think act independently of each other.

Variables that may affect the outcome of an electrophoresis process are the voltage, the gel type and concentration, and other design factors related to electrophoresis setup. We will manipulate these variables in order to fine tune the electrophoresis technique.

Hypothesis:

Based on your gathered information, make an educated guess about what types of things affect the system you are working with. Identifying variables is necessary before you can make a hypothesis.

I think food dies are combination of two or more dies. My hypothesis is based on my observation of making paint that is often done by mixing two or more paints.

Experiment Design:

Design an experiment to test each hypothesis. Make a step-by-step list of what you will do to answer each question. This list is called an experimental procedure. For an experiment to give answers you can trust, it must have a “control.” A control is an additional experimental trial or run. It is a separate experiment, done exactly like the others. The only difference is that no experimental variables are changed. A control is a neutral “reference point” for comparison that allows you to see what changing a variable does by comparing it to not changing anything. Dependable controls are sometimes very hard to develop. They can be the hardest part of a project. Without a control you cannot be sure that changing the variable causes your observations. A series of experiments that includes a control is called a “controlled experiment.”

Introduction: Experimenting Electrophoresis

DNA electrophoresis is exciting but expensive. Hear you will experiment electrophoresis on food dyes. It’s cheaper because you make your own rig and you use a small gel piece and you race food dyes instead of DNA samples. It’s faster: the electrophoresis takes only 15 minutes to get an answer, and no staining and destaining is needed. It’s smaller, so you can do it with less space, less mess and less cost. Overall, these advantages mean you can practice and learn from your mistakes and try your own variations.

Characterizing a compound: The Power of Electrophoresis

Let’s imagine a 100 meter dash. During this race, 8 racers in 8 lanes are separated by their speed. All racers start at the same starting line at the same time, but during the race down the track they separate on the basis of their footspeed.

Electrophoresis is like an electric 100 meter dash. But instead of 1 person running in each lane, think of 10,000 molecules running in each lane. Instead of running on a track, electrophoresis races are run in agarose gels. And instead of being driven by legs, the dyes are driven by electricity.

In this analogy, if the green dye really is a true green, then we would expect there to be a whole bunch of green molecules, and in an “electric race” we’d expect all of them to run the same speed, all in one group.

But if the green dye is really a mix of blue dye and of yellow dye, and if the blue dye molecules runs slower than the yellow dye, then we’d expect the green to separate into two groups, one yellow and one blue, during an electric race.

Should the green dye be the only runner? Designing Appropriate Controls.

Let’s imagine that green is the only runner, and when we run the electrophoresis, it moves as a single band. A skeptical student could ask: how do you know the electrophoresis is even capable of separating the yellow and blue dyes? Maybe the two dyes move at the same speed under the conditions we’re using. Maybe the two dyes when mixed together and exposed to electric current chemically react to form a single compound.

To address these possibilities, let’s run some yellow dye, some blue dye, and some yellow mixed with blue (it looks green). By thinking ahead, by welcoming skepticism, you can design a better experiment. By running a yellow sample next to blue sample, we can test if they move at different speeds or at about the same speed. By running the mixed or “reconstructed” sample of yellow and blue, we can test if electrophoresis can separate yellow and blue, even after they’ve been mixed together and subjected to electricity.

So we have a logic fork: If the green dye is a true green molecule, then we would expect it to “run” as one green band. On the other hand, if the green dye is formed by a mix of blue dyes and yellow dyes, and if the two dyes move at different speeds during the “electric race,” then we expect the green to separate into at least two bands, one yellow and the other blue.

Overview:

Make the electrolyte/buffer.
Make the agarose gel.
Put the electrophoresis rig together.
Prepare the green sample, yellow sample, blue sample, and yellow plus blue mixed sample .
Load the samples into the gel
Race the dyes. Expect about 15 minutes to get an answer.
Ask and observe: Did the green move as a single green band? Or did the green gradually separate into a yellow band and a blue band?

Make the Electrolyte/Buffer.
I have used a Tris-borate buffer (TB). This is what researchers commonly use. It requires Tris base (a powder from a chemical supply company), boric acid from a pharmacy or discount store.

The 10X TB stock solution is made by putting 54 g Tris base and 27.5 g boric acid in water, dissolving it, and bringing the volume to 1.0L.

NOTE: Dilute this 1:10 for use both in making the gel and as the running buffer. For example, 100 ml 10X TB plus 900 ml water to make 1000 ml 1X TB.

Alternate: 0.05M sodium chloride solution. Put 58 g of salt (NaCl) into 500 ml of water and make the volume to 1 liter. The concentration is 1 Molar. Dilute this stock solution 1:20 in tap water to get 0.05 Molar; use the 0.05 M to make gels and for the electrolyte.

Make the Gel.
Try 1% agar in 1X TB.

Place 1g agarose in a 250 ml Erlenmeyer flask, add 100 ml 1X TB, and heat to near boiling.

How to heat the solution?
A microwave oven works well for heating such liquids. A hot plate is an alternative. Cool the hot gel solution to about 55C. Pour it into the plastic container to make a layer about 4-5mm deep. Let the gel cool and harden. The gel can be saved for many weeks as long as you don’t let it dry out.

Alternative to agarose:
Agar as the type used to make agar plates, or agar-agar from a health-food store can be used at 1.0-1.3%.

Make the Gel Rig.
Make sure the adapter is not plugged in.

The AC/DC adapter will have several different adapters at the end of the wire. With a heavy-duty scissors, cut off the adapters, leaving as much of the connecting wires as possible.

Separate the two wires for about 15cm. This will make a kind of wire fork at the end. Strip the insulation off the end of each wire, exposing about 8 cm of wire.

Use the metal binding clips to attach the insulated portion of the wire to the side wall of a second plastic vessel. Leave the exposed 8 cm on the floor of the vessel.

The distance between the wires is adjustable. Later when you’ve cut your large gel into smaller pieces, you can adjust the distance between the wires so that it is just longer than the length of the piece of gel.

Putting the Electrolyte/Buffer in the Rig.

Make sure the adapter is not plugged in. Pour 50-100 ml 1X TB in the plastic vessel. Make sure the two bare wires (electrodes) are immersed. Make sure the two bare wires are not touching each other.

Plug in the adapter and test if current is running through the solution between the electrodes. If the wires are copper, then expect bubbles to form at one wire, and a blue precipitate to form at the other.

UNPLUG the adapter.

Now we have a gel, an electrolyte/buffer and a gel rig.

Cutting the Gel.

The cooled gel covers to a depth of about 5 mm the bottom of the first plastic vessel. How can you use the gel?

One way is to consider it one big gel. Another option is to cut it like brownies into many smaller gels. With a paring knife, cut the gel once down the middle along its long axis. Then cut four times crosswise to get 10 gel pieces. Use the knives to lift any piece out.

How do we load the dye in the gel?

There are many possible solutions to the loading problem. This is the simplest way I know of to load the dyes.
Cut filter paper into long strips 3 mm wide. Dip a filter paper strip into the green dye. Let the dye saturate the lower part of the strip. Hang the strip and let the dye dry for 10 minutes or so. Cut the strip into small rectangles of the same size, about 3×3 mm.

Repeat with clean strips, one for each of the three remaining samples (yellow, blue, and yellow and blue mixed to make a “reconstructed” green). These 3x3mm pieces can be saved indefinitely.

Put one of the gel pieces on a desktop. With a knife make a slit in the gel about 7 mm from one edge. With a tweezers place the dyed filter paper rectangle in the slit.

Make similar slits for the other samples: yellow alone, blue alone, and yellow and blue mixed (it will look green).

When completely loaded, the gel should have four “racers” at the starting line.

Running the Gel

Make sure the AC/DC adaptor is turned off.

1. Place the gel piece containing the dyed paper pieces in the gel rig.

2. Use the metal binder clips to adjust the wires/electrodes so that the wires are just barely wide enough apart so the gel piece can fit between them.

3. Add enough electrolyte/buffer to cover the gel. (Note: use the same solution the gel is made of)

4. Plug in the AC/DC adapter.

5. Again observe the electrodes. Bubbles should form at one, and a blue precipitate at the other (if you’re using copper electrodes.)
6. Which way will the dyes move?

7. In my experience, using an adaptor with a maximum rating of 12 Volts and 300 Amps DC, the dyes move perceptibly within 10 minutes.

8. Let them run until the colors are separated sufficiently to tell whether the green is a true green or a mixture of blue and yellow. The run time depends on the power supply and electrolyte/buffer you use.

Materials and Equipment:

Final list of material may vary based on the changes that you may make in your experiment design.

Set of food colors; this activity is designed for a set with green, yellow and blue($2)
Rubbermaid-type small container with snap-on lid ($2)(4″ x 6″, 10cm x 15 cm)
AC/DC converter, 120 V AC in, 12 V 300mAmps DC out ($10)
agarose or agar (science supply house) or agar-agar (buy at health food store )
Powders to make the electrolyte/buffer: Tris base (science supply house) and boric acid. (0.05M NaCl or phosphate buffer are inferior substitutes as electrolyte.)
forceps or tweezers or spring-loaded hairclips
paring knife or jackknife
filter paper (3 MM or other–needs to absorb the food colors)
paper binder clips made of metal

Cost: Under $30 for the gel rig, agar and dyes.

Results of Experiment (Observation):

Experiments are often done in series. A series of experiments can be done by changing one variable a different amount each time. A series of experiments is made up of separate experimental “runs.” During each run you make a measurement of how much the variable affected the system under study. For each run, a different amount of change in the variable is used. This produces a different amount of response in the system. You measure this response, or record data, in a table for this purpose. This is considered “raw data” since it has not been processed or interpreted yet. When raw data gets processed mathematically, for example, it becomes results.

Calculations:

No calculation is required for this project, however if you do any calculations, write your calculations in your report.

Summary of Results:

Summarize what happened. This can be in the form of a table of processed numerical data, or graphs. It could also be a written statement of what occurred during experiments.
It is from calculations using recorded data that tables and graphs are made. Studying tables and graphs, we can see trends that tell us how different variables cause our observations. Based on these trends, we can draw conclusions about the system under study. These conclusions help us confirm or deny our original hypothesis. Often, mathematical equations can be made from graphs. These equations allow us to predict how a change will affect the system without the need to do additional experiments. Advanced levels of experimental science rely heavily on graphical and mathematical analysis of data. At this level, science becomes even more interesting and powerful.

Analogy:
This is a dye race. Imagine you’re in a blimp over the Olympic track stadium, and below the 100 meter dash is just about to begin. But instead of only one person per lane, imagine 100 people in a lane. In lane one there are 100 people who all run the same speed and who all have on blue shirts; 100 other people in yellow shirts are in the second lane who run twice as fast as the blue-shirts; and 50 people in blue shirts and 50 people in yellow shirts are in the third lane; and 100 green-shirted people in the fourth lane who all run the same speed. The race is a sprint, and you take a snapshot when the first runners cross the finish line. What does the picture look like?

Conclusion:

Using the trends in your experimental data and your experimental observations, try to answer your original questions. Is your hypothesis correct? Now is the time to pull together what happened, and assess the experiments you did.

Possible Errors:

If you did not observe anything different than what happened with your control, the variable you changed may not affect the system you are investigating. If you did not observe a consistent, reproducible trend in your series of experimental runs there may be experimental errors affecting your results. The first thing to check is how you are making your measurements. Is the measurement method questionable or unreliable? Maybe you are reading a scale incorrectly, or maybe the measuring instrument is working erratically.

If you determine that experimental errors are influencing your results, carefully rethink the design of your experiments. Review each step of the procedure to find sources of potential errors. If possible, have a scientist review the procedure with you. Sometimes the designer of an experiment can miss the obvious.