Title: H 2 O that Glows!

Transcription

1 Title: H 2 O that Glows! Gabrielle Powers West Shore Jr. / Sr. High School Abstract: These series of lessons take the idea of Chromatography that is already covered in the Honors and Regular Chemistry class and builds on it to integrate new biotechnology techniques and emerging pathogens. The first lesson gives the background information about techniques used to separate mixtures. Paper chromatography will be performed using markers, as well as the dye obtained from a glow stick. In the second lesson, a video clip will introduce the concept of bioluminescence in some organisms, and how they use the green fluorescent protein to communicate with other organisms in their environment. A Web Quest will follow which will highlight some of the bioluminescent organisms of our world. Finally, all of the lessons will be tied together to reinforce student learning. Using E. coli cells transformed with the Green Fluorescent Protein (GFP) gene from jelly fish prepared by the AP Biology class, the protein will be isolated and the GFP Chromatography Kit from BioRad will be used to analyze the bands. Rationale: The area of biotechnology is expanding and is being heavily relied on for much of the research that is going on today. This research and potential discoveries may have an impact on you or someone you know in the near future. Students may have heard of biotechnology but may not know what it is or think that they are capable of performing some of these same techniques that scientists use in the research laboratories. With the implementation of these technologies, use of the simulations and/or equipment lockers provided by the University of Florida to ICORE participants and their students, these children will have the first hand experience that is relevant to them. This will be highly engaging and open the doors to not only their curiosity and questions, but maybe open their eyes to their potential and their interests they may not have known they had without these experiences. It is our hope as teachers, that our students have the passion and enthusiasm for science that we do. With this introduction to this biotechnology and the amazing science that goes far beyond what we teach them, students will hopefully invest an interest in finding more solutions to problems and what is out there in a really fun and remarkable way. Day 1 3: Separation of Mixtures During this first lesson, the students will view a Power Point to learn about different separation techniques for mixtures and compounds. (Some of these processes used to separate mixtures include decantation, filtration, distillation and paper chromatography. Those used to separate compounds include electrolysis and intense heat.) The students will perform a hands on activity to make sense of one of the processes used to separate mixtures.

2 Assignment: The students will complete the Paper Chromatography Lab Activity which is used to separate the dyes in various colors and brands of markers. In addition, another component will be added where the students will complete a chromatogram of the dyes various colored glow sticks. The students will then take home the reading Creatures that Glow, and answer the questions for homework. Assessment: The students will answer pre lab and post lab analysis questions to show their understanding of chromatography as it s used in the separation of mixtures. These lab reports will be turned in and graded. The reading questions will be turned in and graded. Day 4: Application of Glowing Chemicals to Organisms A connection will be made between the glowing chemicals in glow sticks, and the use of chemicals by bacteria in communication. They will be introduced to many microorganisms of our world, and how they have an impact on their surroundings. The students will complete a Web Quest researching several luminescent organisms of our world. Assignment: The students will view a Power Point (Tiny Organisms, Enormous Effect) to be introduced to the idea of bioluminescence, and learn about the various microorganisms (including various Vibrio species, E. coli and Dinoflaggelates) that have an impact on the environment. They will then watch an 18 minute video with Bonnie Bassler on How Bacteria Talk from Assessment: Students will turn in the Web Quest question sheets to be graded. Day 5 7: GFP expression and isolation from transformed E. coli If the AP Biology teacher has completed the transformation lab of E. coli with the GFP (Green Fluorescent Protein) gene from Jelly Fish, use the E. coli harvested for this portion of the lesson. (If this lab has not been done, a transformation of E. coli with the GFP gene from Jellyfish must be performed first, or already transformed bacteria may be purchased. Either the kit to transform the E. coli or the already transformed E. coli can be purchased from BioRad.) Assignment: The students will then use the GFP Chromatography Kit from BioRad to isolate and analyze the GFP from Jellyfish that was transformed into the E. coli. The process of chromatography will be discussed. Assessment: The students will complete the lab assignment and answer pre & post lab (analysis) questions which will be collected and graded. Extensions Possible/Optional Field Trip:

3 A Day Away Kayak Tours or Haulover Canal Launch, Merritt Island National Wildlife Refuge, FL Adults & Young Adults $34.00 ($38.00 on Saturday); Child $26.00 ($28.00 on Saturday) You can see bioluminescence during the warmer summer/fall months on the waters of the Indian River and Mosquito Lagoon. Move your paddle through the water to see the light green glow of the water. Watch the bright greenish blue speeding rockets swim under your kayak, which are the schools of mullet beneath you. The kayak tour lasts between 1.5 and 2 hours. (For the best results, go when there it is overcast, or when there is little moonlight.) Depending on the Brevard County School District s approval, students will be taken on a field trip to see first hand how bioluminescence occurs in their own backyard. Chromatography: To have another variable for the chromatography lab, students can use multiple brands of markers to see if the various brands of markers use the same dye compounds for each color. Podcasts: Video clips on Bioluminescence in Organisms and/or Green Fluorescent Proteins can be viewed at ww/podcasts9.html#here. Data Collection & Expected Outcomes: A Chapter Test will assess the students knowledge of the processes used to separate mixtures. A set of reading questions will be used to assess the students understanding of what organisms in their world use bioluminescence for. A Web Quest with questions about bioluminescent organisms of our world will be completed and graded. Pre and Post Lab questions will be used to determine the whether or not the student understands the concepts the lab was illustrating. After these lessons the students answers to the analysis (pre & post lab) questions should be well thought out and demonstrate an understanding of chromatography. ICORE / UF Equipment Lockers Needed: Designer Plates Micropipetting Techniques (Unrelated Activity) Antigen/Antibody Immunoassay: Food Allergy Simulation from Dr. Chuck Lawrence (Unrelated Activity) Testing allergy to Casein, Gluten, Red Food Coloring, and Peanuts. Literature Cited: Images (for Power Points): Berger, Melvin, and Damon Hertig. Creatures That Glow: a Book about Bioluminescent Animals. New York: Scholastic, Print. Video How Bacteria Talk :

4 Definitions & Background Information: Mills, C.E present. Bioluminescence of Aequorea, a hydromedusa. Electronic internet document available at Published by the author, web page established June 1999, last updated 11 January Web Quest & Bioluminescent Organisms: season brings glow in the dark mushrooms/ hat_double_as_an_ex.php glow 05.html A Day Away Kayak Tours: Podcasts (for more information on Bioluminescence & GFP): ww/podcasts9.html#here Budget & Budget Justification: Company Item # Quantity Description Unit Price Total BioRad EDU 6 Green Fluorescent Protein Chromatography Kit $ $ ( 8 student work stations) Flinn Scientific AP Ultraviolet Lamp, Hand Held $33.50 $134.00

5 Title: H 2 O That Glows! Key Questions: This lesson will address the following questions: What is Chromatography? What is Bioluminescence? What types of organisms glow, and what are the benefits to glowing? What is Gel Electrophoresis? How are Paper Chromatography and Gel Electrophoresis similar? Science Subject: Chemistry Grade & Ability Level: 9 th 10 th Graders (enrolled in Honors & Regular Chemistry) Key Science Concepts: This lesson will address the following scientific concepts: Paper Chromatography (Identify mobile phase & stationary phase) Separation of Mixtures using Chromatography (Using dyes in markers, and glow sticks) Bioluminescent Organisms (Benefits to glow proteins. How organisms use light to communicate.) Transformation of E. Coli bacteria with GFP from Jelly Fish (E. Coli produced from AP Biology class will be used, or already transformed E. Coli will be purchased.) GFP Protein Chromatography from transformed E. Coli OVERALL TIME ESTIMATE: This series of lessons will be covered over a 7 day period. Day 1 will be the discussion of the Separation Techniques Power Point, background information reading on paper chromatography and Pre Lab questions. Day 2 is the Paper Chromatography Lab. Day 3 will be where the students compare their chromatograms and look at their glow stick chromatogram under the ultraviolet light. The Post Lab questions will also be completed. For homework, students will read Creatures that Glow, and answer questions based on the reading. Day 4 is devoted to background information on bioluminescence and green fluorescent proteins (GFP). A video clip How Organisms Talk at will be played.

6 Day 5 7 students complete the GFP Chromatography Activity (BioRad Kit) from the already transformed E. Coli. LEARNING STYLES: This unit plan will address visual, auditory and kinesthetic learners. The visual and auditory learners will benefit from the video clip, power point and the web quest. The kinesthetic learners will benefit from the hands on Chromatography and Protein Gel Electrophoresis activity. VOCABULARY: Paper Chromatography: a method of separating and identifying the components of a mixture by movement through a two phase (mobile and stationary) system. Immiscible: not able to be mixed. Mobile Phase: the phase that is moving in chromatography. Stationary Phase: the phase in chromatography that is not moving. Capillary Action: the process in which a liquid is able to travel along the surface of a solid, usually against gravity, and is caused by the attraction of the molecules of the liquid to the molecules of the solid Affinity: an attraction for an object Chromatogram: the developed paper containing a pattern of separated chemicals located at different positions created using paper chromatography. Bioluminescence: production and emission of visible light by a living organism resulting from a chemical reaction that converts chemical energy to light energy. The Greek term Bios means living and the Latin term lumen means light. Green Fluorescent Protein: a protein composed of 238 amino acids that glows green under fluorescent light. It is commonly found in the jellyfish Aequorea victoria. LESSON SUMMARY: A power point will be used cover the concepts of separation of mixtures using various procedures. The paper chromatography lab will be used to separate the different dyes of various colored markers as well as the components of different colored glow sticks. An online Video Clip and Web Quest will be used to illustrate the process of chemilumenescence and bioluminescence in a variety of organisms. Finally, the students will complete a hands on activity of purifying Green Fluorescent Protein from the E. coli transformed from the AP Biology class. This GFP Chromatography uses HIC columns is purchased from BioRad.

7 STUDENT LEARNING OBJECTIVES WITH STANDARDS: OBJECTIVES: The student will be able to Identify the methods for separating mixtures versus pure substances. 2. Explain how the process of paper chromatography is used to separate components of mixtures. 3. Identify if there are similarities in the components of various colored markers and glow sticks once they are separated on the chromatograms. 4. Define bioluminescence and give examples of what organisms are bioluminescent and why they use it. 5. Explain how the process of chromatography can be used to isolate the green fluorescent protein from the E.coli bacteria that has been transformed with the GFP gene from jellyfish. STANDARDS: SC.912.N.1.1: Define a problem based on a specific body of knowledge. Pose questions about the natural world, conduct systematic observations, examine books and other sources of information to see what is already known, review what is known, plan investigations, use tools to gather, analyze and interpret data, pose answers, explanations or descriptions of events, generate explanations, or descriptions of events that describe natural phenomena, use appropriate evidence and reasoning to justify these explanations to others. SC.912.P.8.2: Differentiate between physical and chemical properties and physical and chemical changes of matter. MA.912.S.1.2: Determine appropriate and consistent standards of measurement for the data to be collected in an experiment. LA : The student will organize the information to show understanding or relationships among facts, ideas and events. MATERIALS: ESSENTIAL MATERIALS: (per lab group of 2 4 students) 2 pieces of (5.0 cm x 10.0cm) Chromatography Paper, 2 petri dishes, 1 50mL reagent bottle of Solvent A(isopropyl alcohol), 5 Q Tips or toothpicks. 1 paper towel. Computer Lab (1 computer per student or per pair is ideal.) BioRad Kit: GFP Chromatography from transformed E. Coli (shared between several lab stations of 6 12 students) 1 package of 8 10 Crayola Markers, 4 8 various colored glow sticks. 1 hand held ultraviolet light. (per class) 1 tube of GFP Gene Transformed E. Coli. Centrifuge. SUPPLEMENTAL: Power Point on Separating Techniques, Chromatography Lab Procedure with background information, pre and post lab questions, Creatures that Glow reading with questions, Power Point on Tiny Organisms Enormous Impact, Video Clip on How Organisms Talk, Web Quest worksheet, GFP Chromatography Lab Procedure.

8 BACKGROUND INFORMATION: Chromatography see attached Chromatography Lab Vibrio, E. coli and Dinoflagellates see Tiny Organisms, Enormous Effects Power Point attached. Bioluminescence in Aequorea victoria See attached document. ADVANCE PREPARATION: In advance the teacher should check the listed websites for the video clip and web quest to be sure they are not blocked by the school server so their students can access them. Chromatography paper (5.0cm x 10.0cm) should be measured and cut two papers for each lab group. Solution A (isopropyl alcohol) for the Paper Chromatography lab put into the 50ml reagent bottles. PROCEDURE AND DISCUSSION QUESTIONS WITH TIME ESTIMATES: Day 1: 1. Show the Power Point on Separation Techniques. (attached) ~ 15 minutes 2. Have the students read the background information on Paper Chromatography. (attached) ~ 5 minutes 3. On their own paper, the students should set up their lab paper by writing out the Title, Problem, coming up with a hypothesis (in if/then form) and write out and answer the Pre Lab questions. ~ 20 minutes 4. If time allows, discuss the answers to the pre lab questions: ~ 10 minutes Pre Lab Questions: Write out the following questions and answer them on your paper. 1. Define Chromatography. Answer: a technique used for separating mixtures of compounds through a two phase system 2. Name two phases found in chromatography. Answer: mobile phase and stationary phase 3. According to the textbook, what is a solvent? Answer: the dissolving medium in a solution 4. What is the mobile phase in this lab? Answer: the solvent (isopropyl alcohol) 5. What is the stationary phase in this lab? Answer: the chromatography paper (more specifically, the water molecules attached to the paper) Day 2:

9 1. Students will complete the Paper Chromatography Lab following the lab procedure. (attached) ~ 40 minutes 2. While the students are waiting for the chromatograms to develop, they should begin writing down the Post Lab questions on their lab papers. ~ done during lab 3. Be sure to designate an area of your classroom for the students to put the developed chromatogram (standing up on a paper towel) to sit overnight. 4. Students will begin writing out and complete answering the Post Lab questions. ~10 minutes Day 3 1. This is the second day of the Paper Chromatography Lab where the students will analyze their chromatograms. Tell the students to look for similarities in their two chromatograms (Crayola markers vs. glow sticks). ~ 5 minutes 2. The students will look at their glow stick chromatograms under the ultraviolet light. ~ 5 minutes 3. Students will finish writing out and complete answering the Post Lab questions. ~20 minutes 4. If time allows, review answers to Post Lab questions (below) ~ 20 minutes 5. Homework: Send students home with Creatures that Glow reading, and have them answer the questions (document attached, and answers are provided ). Post Lab Questions: Write out the following questions and answer them on your paper. 1. How does the solvent travel up the paper? Answer: by capillary action caused by the attraction of the molecules of the liquid to the molecules of the solid. 2. Explain how the separation of mixtures occurs. Answer: the physical interaction of each compound in the mixture with the solvent and with the water molecules attached to the paper determines the distance the dye travels. 3. What happened to the ink spots as the chromatogram develops? Why? Answer: they dissolve in the solvent, and travel up the paper by capillary action. They will adhere to the paper (the water molecules attached to the paper) when the attraction for the water molecules becomes greater than the ability for it to be dissolved in solution. This will leave the components of the mixture separated at different heights on the chromatogram. 4. If one dye moves up farther than another, which one dissolves more readily in the solvent? Answer: Substances that dissolve better in the solvent will travel farther than substances that have a higher attraction for the water attached to the paper. 5. Which marker(s) and glow stick(s) (specify original color and marker/glow stick) are probably made of only one compound? How can you tell? Answer: (answers may vary.) If the marker and/or glow stick is made up of only one compound, there will only be one spot of color or dye on the chromatogram along the column that corresponds to that color. 6. How can you tell which marker(s) and glow stick(s) (specify original color and marker/glow stick) are made up of more than two compounds? Which are they? Answer: (answers may vary.) If the marker and/or glow stick is made up of more than two compounds there will be more than two marks of color/dye on the chromatogram along the column that corresponds to that color. 7. Compare the two chromatograms. Did both markers and glow sticks use the same combination of dyes to make the same colors? How can you tell?

10 Answer: (answers may vary.) There should be a similarity in the colors of the markers and glow stick color components. This is seen by having the spots on the two chromatograms in similar locations. Day 4: 1. Collect Creatures that Glow reading and the student s answers to the questions. 2. The students will view a Power Point on Tiny Organisms, Enormous Effects (attached) to be introduced to a few microorganisms in their world and the effects they have on people. ~ 10 minutes 3. The students will be introduced to the concept of bioluminescence (and the green fluorescent protein) by watching a video clip How Organisms Talk at: ~20 minutes 4. The students will then begin a Web Quest (attached document). ~ 30 minutes *They can finish these Web Quests for homework, or you can allow more class time another day.* Day 5 7: 1. The students will read through and become familiar with the procedure for the GFP Chromatography (ordered through BioRad) from E. coli transformed with the GFP gene. Discuss the procedure. ~ 10 minutes 2. The students will then complete the GFP Chromatography Lab Procedure (BioRad Kit) from the already transformed E. Coli. ~ 3 classes of 40 minutes 3. The students will then analyze their data and try to locate the extracted protein band using an ultraviolet light. ~ 10 minutes 4. Discuss the significance of the results of the lab. ~ 10 minutes **I have not done this lab before, so these are estimations on time limits** ASSESSMENT SUGGESTIONS: Objective 1 will be assessed during the Chapter Test. Objective 2 & 3 will be assessed through the Pre and Post Lab questions of the Paper Chromatography Lab which addresses the student s comprehension of the concept of separation of mixtures. Answers will be graded with answer key provided. Objective 4 will be assessed by the reading questions based on Creatures That Glow, which introduces the idea of bioluminescence and what organisms use bioluminescence for. Grade answers with answer key provided. The Web Quest will also be used to assess mastery of Objective 4. These will be graded using the answer key provided.. Objective 5 will be assessed by the GFP Chromatography Lab questions. *I have not done this lab before to know what probing questions I can ask based on the background and procedure information provided with the kit.*

11 EXTENSIONS: ACTIVITIES: Chromatography: Add another variable and have the students make a third chromatogram to analyze another brand of markers (Roseart) to see if there are similarities or differences in the different dyes that make up the different colors between brands of markers. Field Trip: A bioluminescent kayak tour at A Day Away Kayak Tours in Titusville, FL. ( This could be an organized field trip or just an extra activity you tell students to go to, or meet you at. Podcasts: View a video clip on Bioluminescence and/or Green Fluorescent Proteins at ww/podcasts9.html#here LITERATURE: Creatures that Glow: A Book About Bioluminescent Animals by Melvin Berger RESOURCES/REFERENCES: Images (for Power Points): Berger, Melvin, and Damon Hertig. Creatures That Glow: a Book about Bioluminescent Animals. New York: Scholastic, Print. Background Information: Mills, C.E present. Bioluminescence of Aequorea, a hydromedusa. Electronic internet document available at Published by the author, web page established June 1999, last updated 11 January home.html Web Quest Additional resources used for Web Quest: season brings glow in the dark mushrooms/ at_double_as_an_ex.php glow 05.html Online Video Clip: How Bacteria Talk with Bonnie Bassler: Extension Activity: Field Trip

12 Podcasts (for More information on Bioluminescence & GFP): ww/podcasts9.html#here

13 Bioluminescence and Green Fluorescent Protein in Aequorea victoria Bioluminescence occurs when a living organism produces and emits visible light, which is a result of a chemical reaction that converting chemical energy to light energy. The term comes from the word Bios in Greek meaning living and the Latin lumen meaning light. This process of bioluminescence occurs in the Aequorea victoria of Puget Sound, Washington. These jellyfish are found in the northeast Pacific Ocean ranging from the Bering Sea to southern California. Several species names have been given to Aequorea due to some apparent physical differences in size, but it is unknown whether or not some of these organisms are the same as or a different species than the A. victoria. These adult jellyfish are very small in comparison to other jellyfish commonly thought of. They only grow to be between 5 to 10 centimeters, and they only live for 6 months or less. By mid-autumn, the whole A. victoria population dies and then is regenerated the following spring by the polyp colonies that have settled and attached themselves to shells and other hard surfaces on the ocean floor. These polyp colonies were created by the Aequorea daily spawning of eggs or sperm; when the eggs are fertilized they develop into larva and swim/settle to the bottom to grow the hydroid colony. The A. victoria is a commonly studied jellyfish as it is fairly easy to grow in the laboratory. The luminescent protein aequorin causes the A. victoria to produce a light that is bluish in color. In these jellyfish, there is another luminescent protein called a green fluorescent protein (GFP). The GFP is a protein that contains 238 amino acids. When the aequorin and GFP are paired together and light is emitted, the GFP causes the light to appear a greenish color. These luminescent protein molecules have been isolated and analyzed in these jellyfish.

14 A common misconception is that the whole jellyfish glows all over. This is not the case. In fact, these organisms are only bioluminescent around the rim of the bell, appearing as a string or ring of lights. The organism itself is colorless and transparent, but some printed photographs have been altered to show a false all over blue or green bioluminescent glow. In fact, aside from the green ring of luminescence around the bell, the rest of the organism would be invisible in the dark. The Aequorea can be stimulated to bioluminesce. These organisms do not sting, so if you pick one of them up (at nighttime) and gently shake them in your hand, you will briefly see the Green Fluorescent Protein in action as a ring of green bioluminescence. It is not yet understood what these jellyfish use bioluminescence for. When introduced to cells of other organisms, the GFP can serve as a convenient marker for gene expression, as well as fluorescent tag for certain proteins. This transformation of the GFP gene is commonly performed with E. coli, as well as many other organisms, in research laboratories. Cellular imaging using optical microscopy of these fluorescent markers fused to protein and enzyme targets can occur under low light for many hours at a time. This enables researchers to witness the cellular processes taking place, track down and identify proteins or organelles of interest, and expose finite details of the structure and function of various components of the cell. Citation: Mills, C.E present. Bioluminescence of Aequorea, a hydromedusa. Electronic internet document available at Published by the author, web page established June 1999, last updated 11 January scentproteinshome.html

15 Creatures that Glow By Melvin Berger Creatures that glow live almost everywhere. You can see them flying through the air, crawling on the ground, and swimming in the sea. Creatures that glow are called bioluminescent. The word has three parts. Bio means life. Lumin means light. Escent means being. Bioluminescent creatures are living beings that give off light. But what kind of light do they make? The glowing creatures light is not like the light of light bulbs, candles, the sun or fire. This is hot light that gives off heat. Creatures that glow make cold light that does not give off heat. Most bioluminescent creatures make their own light. They are born with certain chemicals. These chemicals mix together in their bodies. When this happens, the creatures begin to glow! Other creatures that glow cannot make their own light. Instead, they get their light from glowing bacteria. Bacteria are tiny beings. Certain bacteria make their own light. Many live inside animals. Their light shines through the animals skin. This makes the animals glow. Not all bioluminescent creatures are the same. Some glow with one color. Others glow with many colors. Some lights flash on and off. Others shine with a steady light. Some use their light to find mates. Others use their light to frighten enemies. Some glow to light their way. Others glow to attract prey. Imagine what life would be like if you could glow! Fireflies Fireflies are also called lightning bugs. But fireflies are not bugs. Fireflies are not even flies! Fireflies are beetles. Most are about as long as one of your fingernails. During the day they look brown and gray. But after sunset the back ends light up like little lamps! Fireflies glow with a yellow-white light. They flash the light on and off. Fireflies use their light to find mates. The male flashes his special signal. The female flashes her answer. There are many kinds of fireflies. One kind is called a click beetle. People sometimes call them fire beetles. Most click beetles glow with three bright lights. Two round yellow lights are near the front. A red light glows in back. Can you guess why click beetles are sometimes called auto bugs? Click beetles do not flash their lights on and off. Instead they keep a bright steady light. Click beetles live in the jungle. People catch these beetles. They put them in see-through bags. And they tie the bags around their ankles and wrists. The glow from the bags helps light their way along jungle paths at night. People also keep click beetles in cages to light their homes. Glowworms Most glowworms look like short, fat worms. But they are not worms. Many glowworms are an early stage in the life of some insects. These insects begin life as an egg. Then the egg hatches, and out crawls a grub. We call this grub a glowworm. Glowworms can glow with a bright light. In time, the grub develops into a fully grown insect.

16 One kind of grub glowworm lives in caves. It builds small nests that hang from the caves roof. The tiny glowworms group in thousands on the roof. The light they make together is bright enough to read by. These grub glowworms eat small insects. They catch the insects in a special way. They spin their saliva into long, sticky threads. The threads dangle from their nests. The glowworms shine their bright lights. Some insects fly toward the light. They get caught in the hanging threads. Quickly the glowworms slide down the threads. Crunch! The glowworms gobble up the insects! Railroad Worms Railroad worms are also glowworms, but they are the adult stage of certain insects. Railroad worms are adult beetles without wings. Railroad worms are hard to find. By day they don t glow. Often they hide under logs or rocks. But at night the railroad worms come out. They start to glow if they are touched. They also glow if they hear a loud sound. And they glow with different colors! On the head of each railroad worm is a bright red light. It looks something like a train headlight. Eleven yellow-green lights line each side of the body. These lights look like the windows of a night train. Usually the worms curl up tightly. At night they look like trains going around a curve. Centipedes Centipedes look like thin worms with many, many legs. Certain kinds can make their own light. Their bodies don t glow. Instead, they give off a poison that glows. Some bioluminescent centipedes are blind. Therefore, they cannot see the light they make. So these centipedes can t use their light to find mates or food. Blind centipedes that glow use their light in another way. They use it to scare away enemies. Suppose an enemy attacks a centipede. The centipede squirts its glowing poison over the enemy. The poison burns like fire, and it smells very bad. The enemy flees. Later the enemy may spot another centipede s glowing poison. What happens? The enemy remembers this poisonous glow. It is a warning to get out of the way fast! Jellyfish Jellyfish are sea animals. They look like open umbrellas. The smallest jellyfish are the size of your thumb. The biggest are as wide as a rowboat. A few kinds of jellyfish give off light. Jellyfish make their own light. One type has a few light rings around its body. Another has a border of light spots. They use their light to find food. Some small prey see the light of the jellyfish and swim toward the light. Long, thin threads hang from the jellyfish s body. The threads sting the prey that get too close. Then the jellyfish pulls the prey into its mouth. Hatchet Fish Hatchet fish look just like their name. They are small and silvery, and they are shaped like small hatchet blades. These strange creatures live near the ocean bottom. Here it is very, very dark, but the hatchet fish has rows of pink lights along the bottom of its body.

17 Hatchet fish use their light to catch prey. This is how it works: a hatchet fish rises to the surface of the ocean to chase something to eat. The rows of pink lights make the fish sparkle as it swims along. It blends in with the water s glistening light, made by the sunlight shining from above. The other fish do not see the hatchet fish. Some swim too close. The hatchet fish opens its huge mouth. Gulp! It swallows the unlucky prey. Squid Squid are sea animals that have no bones. Their bodies are very soft. Many make their own light. They use their lights to help them find food. The lights also help squid find one another. The deep-sea jeweled squid is very special. It glows with many colors. The middle lights are blue, while the outside lights are gray. Eight lights shine on the squid s underside. The front lights are red, the middle lights are blue and the back lights are white. The firefly squid is another squid that glows. It has many small lights covering its body. During the day, the creature looks dull and spotted. But at night it glows with many bright blue lights. The bodies of other types of squid do not glow. But they can squirt out a liquid that glows blue! If the squid feels threatened, it squirts the glowing liquid into the dark water. The liquid blinds enemies temporarily while the squid swims safely away. Angler Fish Angler fish live in the deepest part of the ocean. One is small enough to fit in your hand. But watch out! Angler fish have great big mouths and many sharp teeth line their jaws. A long rod sticks up from the head of the angler fish. The rod looks like a tiny fishing pole. At the end of the pole is a blue-green light. The light dangles in front of the fish. As an angler fish swims, it flashes its light. The flashes attract many small sea animals. These animals come close and the angler fish opens its mouth. Snap! In goes dinner. Flashlight Fish Flashlight fish are sometimes called lantern eyes. Both names come from the way these fish look. Under each eye is a small bag of skin. The bags glow with a bright blue light. Flashlight fish glow but do not make their own light. Their light comes from billions of tiny, glowing bacteria. The bacteria live inside the fish s eye bags. A bright blue glow shines through the skin of the flashlight fish s eye bags. It makes the bags look like lit lanterns. The bacteria give off a steady glow. But what happens when the flashlight fish wants to shut off the light? It covers each bag with a thick fold of skin like an eyelid. This cuts the light off completely. Flashlight fish use their light in many ways: to help them see where they are going, to help them search for food, to help them find mates, and even to help them talk to each other. The light also protects the flashlight fish from enemies. Big fish often try to catch flashlight fish. The flashlight fish try to confuse their attackers. They suddenly shut off the light. Or the flashlight fish start to zigzag, blinking their lights on and off rapidly. After a while, the enemies give up trying to catch the flashlight fish. They swim away to look for other food.

18 There are thousands of other bioluminescent creatures. They vary widely in shape and color. They live in most climates on Earth, and they can be found in all levels of the ocean. So keep an eye out. You may see some Creatures That Glow. Based on your reading, answer the following questions in the space provided. 1.) What does the term bioluminescent mean? 2.) What do fireflies use their light for? 3.) Why do people catch click beetles? 4.) What do glow worms use their light for? 5.),,, use their bioluminescence to find food, whereas some use their bioluminescence for protection by squirting their enemies with glowing poison. 6.) Flashlight fish use their bioluminescence to ANSWER KEY: 1.) What does the term bioluminescent mean? Answer: living things that give off light 2.) What do fireflies use their light for? Answer: to find mates 3.) Why do people catch click beetles? Answer: They give off a steady glow/light. They put them in clear bags and tie the bags around their ankles so they can see when they are walking through the dark woods. 4.) What do glow worms use their light for? Answer: to attract insects to fly into and get trapped in their sticky, hanging threads, so the glow worms can then eat the insects. 5.) _Jellyfish_, _Hatchet fish_, Squid_, Angler Fish_ use their bioluminescence to find food, whereas some Centipedes use their bioluminescence for protection by squirting their enemies with glowing poison. 6.) Flashlight fish use their bioluminescence to help them see, search for food, find mates, and talk to each other. They also use their light to protect them from enemies by confusing them by blinking their light on and off and swimming away._

19 Paper Chromatography Objectives Separate mixtures of dyes, using the technique of paper chromatography. Identify similarities of compounds contained in various colored markers. Compare formation of various colored markers to components of various colored glow sticks. Introduction Paper Chromatography is a technique used for separating mixtures of compounds through a two-phase system. It is a powerful and versatile method used widely in chemistry and in the biological sciences. Chromatography can also be used to identify unknown substances. All of the several types of chromatography employ two different immiscible phases, or substances that are not able to be mixed with each other. In the case of paper chromatography, one of the phases is moving, the mobile phase, and the other is not, the stationary phase. In paper chromatography, a solvent moves from one end of a piece of paper to the other end, as the paper absorbs it. The solvent is the mobile phase because it is moving, and the paper is the stationary phase. (In reality, there are water molecules attached to the paper that serve as the stationary phase.) A mixture of chemicals can be separated using paper chromatography. A small amount of mixture to be separated is placed near the edge of an absorbent paper. That edge of the paper is wetted with solvent. The solvent travels up the paper by capillary action, carrying the mixture with it. Capillary action is the process in which a liquid is able to travel along the surface of a solid, usually against gravity, and is caused by the attraction of the molecules of the liquid to the molecules of the solid. Separation of the components of a mixture occurs because different chemicals in the mixture travel different distances. The physical interaction of each compound in the mixture with the solvent (the mobile phase) and with the water molecules attached to the paper (the stationary phase) determines the distance it travels. Substances that dissolve more readily in the solvent will move farther than substances that have a higher affinity, or attraction, for the water attached to the paper. When the solvent has moved the entire length of the paper, the paper is removed from the solvent and dried. Once developed, the paper containing a pattern of different chemicals located at different positions on the paper is referred to as a chromatogram. The various chemicals visible on the chromatogram can often be identified by their positions and/or their colors. If the mixture contains colored compounds, each different compound will appear on the chromatogram as a colored spot or a streak in a particular place. The color and location of each compound can be used as a basis for identification. The color and location of unknown compounds separated under specific chromatographic conditions can be matched with the color and location of known compound subjected to the same conditions. Pre-Lab Questions: Write out the following questions and answer them on your paper. 1. Define Chromatography. 2. Name two phases found in chromatography. 3. According to the textbook, what is a solvent? 4. What is the mobile phase in this lab? 5. What is the stationary phase in this lab? Purpose: In this lab you will use the technique of paper chromatography to separate the colored dyes in a various colors of markers and glow sticks.

20 Hypothesis: (The student writes.) Safety: Wear safety goggles for this lab experiment. Equipment: 2 pieces of 5.0cm by 10.0cm chromatography paper, 8-10 Crayola markers, various colored soft plastic glow sticks, 5 Q-Tips or toothpicks, scissors, pencil, mini stapler, Petri dishes, and Solvent A (isopropyl alcohol). Procedure: Part I: Setting up the Chromatograms 1. Obtain two pieces of pre-cut chromatography paper from your teacher. 2. Mark the two pieces of chromatography paper as follows. Use the template below as a guide. Use only pencil to write on paper. 3. Set up a data table on your paper that looks exactly like this chromatogram. 4. Obtain a set of Crayola markers and draw a small dot of ink from a red pen on the first pencil dot on one piece of chromatography paper. Continue placing colored dots on the pencil dots in the same order as the visible spectrum: red, orange, yellow, light/dark green, light/dark blue,violet, (ROYGBIV). Place other colors such as pink, grey, brown and black at the end. Record on your lab paper the number, color and marker. 5. Obtain a variety of different colored soft plastic glow sticks. Snap the glow sticks so the chemicals begin to mix. 6. Carefully cut the open the glow sticks. Using a Q-Tip or a toothpick, obtain a very small amount of dye from the glow stick and place it on the first mark on #1 of your second chromatogram. Placing the colors in the same order as your first chromatogram, put one spot of dye from the various glow sticks on each numbered location. Record number, color and glow stick on your lab paper. 7. Staple the chromatograms into cylinders, with the colored dots facing out. Be careful not to allow the edges to touch one another (see Figure 1.1). Part II: Developing the Chromatograms 8. Put enough Solvent A (isopropyl alcohol) into the bottom of the Petri dish, but not so much that it is above the pencil line on your chromatograms that the dots were placed on. (see Figure 1.2) 9. Place cylinders into their own Petri Dishes, and begin to record observations.

21 10. Take the chromatograms out of the Petri Dishes when the solvent line is no more than 1cm from the top of the chromatogram. 11. Draw a line on the chromatogram to mark where the solvent line was when you removed the chromatogram from the Petri Dish. Figure 1.1: Stapled cylindrical chromatogram with the dye dots facing outward. Figure 1.2: Stapled chromatograms in Petri Dish with the solvent below the pencil & dye dot line on the chromatogram.

22 12. Place developed chromatograms standing on a paper towel in the area designated by your teacher. Let them dry overnight in order to analyze the following day. Part III: Next Day 13. Obtain your dry, developed chromatograms from the designated area and bring them back to your lab station. 14. Use an ultraviolet light to analyze your glow stick chromatogram. 15. Compare your two chromatograms to identify similarities in compounds. 16. Be sure to staple your chromatograms, and both lab papers for you and your partner, after you complete your post lab questions. Post-Lab Questions: Write out the following questions and answer them on your paper. 1. How does the solvent travel up the paper? 2. Explain how the separation of mixtures occurs. 3. What happened to the ink spots as the chromatogram develops? Why? 4. If one dye moves up farther than another, which one dissolves more readily in the solvent? 5. Which marker(s) and glow stick(s) (specify original color and marker/glow stick) are probably made of only one compound? How can you tell? 6. How can you tell which marker(s) and glow stick(s) (specify original color and marker/glow stick) are made up of more than two compounds? Which are they? 7. Compare the two chromatograms. Did both markers and glow sticks use the same combination of dyes to make the same colors? How can you tell?

23 ANSWERS: Pre-Lab Questions: Write out the following questions and answer them on your paper. 1. Define Chromatography. Answer: a technique used for separating mixtures of compounds through a twophase system 2. Name two phases found in chromatography. Answer: mobile phase and stationary phase 3. According to the textbook, what is a solvent? Answer: the dissolving medium in a solution 4. What is the mobile phase in this lab? Answer: the solvent (isopropyl alcohol) 5. What is the stationary phase in this lab? Answer: the chromatography paper (more specifically, the water molecules attached to the paper) Post-Lab Questions: Write out the following questions and answer them on your paper. 1. How does the solvent travel up the paper? Answer: by capillary action caused by the attraction of the molecules of the liquid to the molecules of the solid. 2. Explain how the separation of mixtures occurs. Answer: the physical interaction of each compound in the mixture with the solvent and with the water molecules attached to the paper determines the distance the dye travels. 3. What happened to the ink spots as the chromatogram develops? Why? Answer: they dissolve in the solvent, and travel up the paper by capillary action. They will adhere to the paper (the water molecules attached to the paper) when the attraction for the water molecules becomes greater than the ability for it to be dissolved in solution. This will leave the components of the mixture separated at different heights on the chromatogram. 4. If one dye moves up farther than another, which one dissolves more readily in the solvent? Answer: Substances that dissolve better in the solvent will travel farther than substances that have a higher attraction for the water attached to the paper. 5. Which marker(s) and glow stick(s) (specify original color and marker/glow stick) are probably made of only one compound? How can you tell? Answer: (answers may vary.) If the marker and/or glow stick is made up of only one compound, there will only be one spot of color or dye on the chromatogram along the column that corresponds to that color. 6. How can you tell which marker(s) and glow stick(s) (specify original color and marker/glow stick) are made up of more than two compounds? Which are they? Answer: (answers may vary.) If the marker and/or glow stick is made up of more than two compounds there will be more than two marks of color/dye on the chromatogram along the column that corresponds to that color. 7. Compare the two chromatograms. Did both markers and glow sticks use the same combination of dyes to make the same colors? How can you tell? Answer: (answers may vary.) There should be a similarity in the colors of the markers and glow stick color components. This is seen by having the spots on the two chromatograms in similar locations.

24 Classification of Matter Mixtures a physical blend of two or more substances that retain their individual identities and properties. Identified by their composition Heterogeneous Homogenous Pure substances a single, homogeneous substance in a fixed composition. Identified by their component(s) Elements Compounds

25 Mixtures Heterogeneous not uniform throughout. Homogeneous completely uniform throughout. (a.k.a. solutions)

26 Separation of Mixtures Mixtures can be separated by: Filtration Distillation Paper Chromatography Decantation

27 Pure Substances Are different from mixtures: Any sample from a pure substance has exactly the same physical/chemical properties. Every sample of pure substance has exactly the same composition.