Separating Pigments Using Paper Chromatography

Our latest unit of study in biology class has been that amazing process on which we all depend- photosynthesis!  Of course, we covered the basics: that photosynthesis is a chemical reaction done by certain organisms allowing them to use the energy of sunlight to convert carbon dioxide and water into glucose and oxygen.  We reviewed the chemical equation for photosynthesis and emphasized the reactants needed for the reaction (carbon dioxide, water, and sunlight) and the products produced by the reaction (glucose and oxygen).  We discussesd the structure of chloroplasts, the organelles in cells where photosynthesis takes place.  We also began to delve into the two stages of photosynthesis, the light dependent reactions and the light independent reactions (also known as the Calvin Cycle).

We begin the unit by talking about the importance of energy for living things- we can't live without it!  Where does our energy come from?  Our food!  Many students already know that some organisms are able to make their own food while other organisms (like us) have to consume other organisms or products of other organisms for food.  We learned that autotrophs (aka producers) are organisms that can make their own food using processes like photosynthesis and chemosynthesis, and that heterotrophs (aka consumers) are organisms that must consume food.  Learning these terms was a nice way to recall our science prefixes and suffixes from the beginning of the year- we remembered that auto means "self," hetero means "different," and troph means "eat or consume." 

One lab that we performed during this unit focused on the pigment that is at the center of photosynthesis, chlorophyll.  Chlorophyll is a green pigment found in the thylakoids, which are little disk-shaped structures inside chloroplasts.  While chlorophyll is responsible for the green color in leaves of plants, its more important role is to absorb the energy from sunlight that drives the reactions of photosynthesis.  The lab introduced students to a scientific technique called chromatography.  There are several different forms of chromatography, but we completed a simple kind called paper chromatography.  In our lab, paper chromatography allowed various pigments in different mixtures to be separated as the pigment mixtures moved along the paper.  Those that were more soluble in our solvent, acetone, moved further up the paper than those pigments that were less soluble in acetone.

Students began by testing out a Mr. Sketch marker.  Some groups tested black markers and some groups tested brown markers, since we hypothesized that these colors would have the most different pigments.  Students placed a dot at the bottom of a piece of chromatography paper and then placed the strip of paper in a test tube with acetone.  As the acetone moved up the paper due to capillary action, it carried the various pigments with it and separated them out on the paper strip.  Students then set up four more strips of chromatography paper with unknown food coloring mixtures.  Their goal was to see if they could determine which colors were mixed based on the pigments that separated on their chromatography strip.  Many got good results and were successfully able to identify the unknown colors in each mixture. 

A student adds a food coloring mixture to a strip of chromatography paper.

Examples of separation of pigments from markers and food coloring mixtures

In the second part of the lab, students were tasked with extracting the chlorophyll pigment from spinach leaves.  Students were given a few leaves of spinach, along with some rocky sand and some acetone, and began to grind the leaves using a mortar and pestle to attempt to extract the chlorophyll.  Once they had a small amount of liquid extract, they poured it into a test tube and placed a strip of chromatography paper down into the extract.  While some worked better than others, most students were able to see various shades of green chlorophyll pigment on their chromatography strip as the liquid extract moved up the paper.  Some even saw some yellow pigment toward the top!  

Students grind up spinach leaves in an attempt to extract chlorophyll pigment!

The ground up spinach- you can see some of the liquid chlorophyll extract!

A good example of chlorophyll pigment separation on a chromatography strip!

Observing Osmosis in Egg Cells

Another fun and interesting activity we performed this month was observing the process of osmosis in eggs.  Students learned that osmosis is the diffusion of water through a membrane (like the cell membrane) from an area of high water concentration to an area of lower water concentration.  We discussed how different types of solutions can affect the process of osmosis causing water to move into cells or out of cells.  Students learned the terms isotonic, hypotonic, and hypertonic to describe the solutions in which cells may be immersed.

We learned that when a cell is in an isotonic solution, it is at equilibrium with its environment- in other words, the water and solute concentrations inside and outside of the cell are equal.  Although there is still some movement of water molecules across the cell membrane, about the same amount of water is moving into the cell as is moving out, so the cell maintains its size and shape.

When cells are in hypotonic solutions, they are surrounded by fluid that has a high water concentration and a low solute concentration.  Because water diffuses from an area of higher concentration to an area of lower concentration, this causes water to diffuse into the cell and the cell increases in size.  If too much water diffuses into the cell, it could burst!  I tell students to remember this because a hypOtonic solution causes the cell to swell up like a big fat letter O!

On the other hand, when cells are in hypertonic solutions, they are surrounded by fluid that has a high solute concentration and a low water concentration.  This causes water to move out of the cell, which will cause the cell to shrivel and possibly dehydrate.  I tell students to remember this because a hypeRtonic solution causes the cell to shrivel up like a Raisin!

We did a lab activity involving egg cells to demonstrate how much these different solutions can affect cell size and shape.  To begin, each lab group was given one regular chicken egg from the grocery store.  Students measured the mass of the egg using a triple beam balance and the circumference of the egg by wrapping a string around the thickest part of the egg and then measuring the length of the string using a metric ruler.  After recording their results, students placed their egg into a plastic cup and added enough vinegar to the cup to completely cover the egg.  They made a prediction about what would happen to the egg overnight, and then left it to sit in vinegar until class the next day.

Our eggs in vinegar.  The acid of the vinegar dissolved the shells of the eggs, allowing osmosis to occur through their more flexible membranes.

The next day, students removed their egg from the cup, rinsed the egg, and rinsed the vinegar out of the cup.  They made some observations about the state of the egg- many were surprised to find that the egg was soft- the vinegar had almost completely dissolved the shell of the egg!  Students again measured the mass and circumference of the egg to see if those measurements had changed from yesterday.  Most groups found that their egg had gotten larger- water had moved into the egg by osmosis!

After taking and recording these measurements, students placed their egg back into the cup and this time covered it with corn syrup.  They made a prediction about what would happen to the egg soaking in corn syrup overnight and then left it until the next day.  What we found the next day really surprised a lot of students.  The eggs were pretty deflated, like a playground ball with all the air let out of it.  When measurements of mass and circumference were taken, students found that the egg had lost both mass and circumference.  We realized that the corn syrup was a hypertonic solution to the inside of the egg, and water had moved out of the egg by osmosis, causing the deflation!

An egg that has been deflated by a hypertonic corn syrup solution!

Students placed the eggs back into their cups for one more overnight soaking.  This time, eggs were covered with distilled water for 24 hours.  Students made predictions and left their eggs one more time.  The next day, they were excited to see that the eggs had re-gained their regular shape, and in fact were a bit bigger than normal.  Measuring mass and circumference confirmed that the eggs were the largest they had been throughout the experiment.  We realized that the distilled water had been a hypotonic solution to the inside of the egg cell, and so a lot of the water had moved into the egg!

An egg that has been swollen with water by a hypotonic distilled water solution!

I love how this lab reinforces the concepts of osmosis and allows students to see osmosis at work.  We discussed some real-life situations that relate to osmosis, such as plants on the side of the road dehydrating when salt is used to treat icy roads, or fruit splitting and being ruined by large amounts of rain.  It has been helpful to have the egg lab to refer back to when I am reviewing these concepts with students- because it was so memorable, it gives them a good frame of reference for osmosis!  

Cell Membranes are Selectively Permeable!

It's been awhile since I last posted, but we've been quite busy in biology class since then.  Now that the end of the second marking period has come and gone, I have a little time to catch up on what we've been doing so far this January...

Our main topic this month has been how the cell membrane allows cells to maintain homeostasis- a state of balance between a cell and its environment.  We talked about the molecules that make up the cell membrane, including phospholipids, proteins, and carbohydrates.  We discussed how these components allow the membrane to be strong, yet flexible, as well as selectively permeable, meaning that it allows some materials to pass through it into or out of the cell while other materials are not able to pass through it.

We did a short lab activity to demonstrate the selectively permeable aspect of the cell membrane.  Students set up a 2 beakers with different solutions.  The students measured water into one beaker and added several drops of iodine to the water, turning it a dark brown color. Students then mixed two spoonfuls of starch with water in a second beaker.  They then poured about half of the starch solution from the beaker into to a ziploc baggie.  They placed the baggie with the starch solution into the first beaker with the iodine water and let it sit overnight.  I reminded students of something we learned in a lab earlier in the year- that iodine is a starch indicator and will turn a black/purple color in the presence of starch, and asked them to make predictions about what will happen to the solutions over a period of 24 hours.

A student measures water to add to two beakers.  One beaker will contain an iodine solution, the other will contain a starch solution.

Starch solution- half was poured into the baggie and half was left in the beaker!

The beaker on the left contains starch solution and the beaker on the right contains iodine solution.  Immersed in the iodine solution is a baggie containing starch solution.

The next day, we returned to our two beakers.  Students added a few drops of iodine to the beaker that contained starch solution, just to observe the color change that occurs when starch and iodine come into contact.  They then removed the baggie containing the starch solution from the beaker with the iodine water to observe what had happened over 24 hours.  Students observed that the starch solution inside the baggie had become purple, while the iodine water remained yellowish-brown.  How can we explain these crazy results?!

The starch solution in the beaker turns purple with the addition of iodine- a starch indicator!

In this picture you can see the starch clump in the left corner of the baggie has turned purple.  The iodine solution left in the beaker has not turned purple.  Iodine was able to diffuse into the baggie to turn the starch purple, but starch was not able to diffuse out of the baggie to turn the iodine purple!

Another view of the beaker of iodine solution and the baggie of starch solution

We remembered that cell membranes are selectively permeable- some materials can pass through them, but not everything!  It turns out that the ziploc baggie in this experiment acted in a similar way- iodine molecules are small enough to diffuse through the tiny, tiny holes in the baggie, so over the 24 hour period, they diffused through the baggie and caused the starch solution inside the baggie to turn purple.  However, the starch molecules are too big to diffuse through the baggie, so they were not able to move outside the baggie- therefore, the iodine solution in the beaker did not turn purple.  Kind of cool!