Overview

 Teacher notes on TLC labOverviewStudents will separate the pigments present in plant leaves. They will develop an understanding of the chemical interactions involved in such separation. This information should help students contextualize other biochemical topics throughout the year. Students will also be able to engage in experimental design with significant scaffolding (there are only a few items the students can change). This experience should prepare them for more complex project-based learning later in the year.Biology standards emphasized in this labHS-LS1-5. Use a model to illustrate how photosynthesis uses light energy to transform water and carbon dioxide into oxygen and chemical energy stored in the bonds of sugars and other carbohydrates. HS-LS1-6. Construct an explanation based on evidence that organic molecules are primarily composed of six elements, where carbon, hydrogen, and oxygen atoms may combine with nitrogen, sulfur, and phosphorus to form monomers that can further combine to form large carbon-based macromolecules. NOTE: This lab does not focus on monomers/polymers, but the prelab helps develop an understanding of organic molecules. Such an understanding is important for connecting intermolecular interactions with biological polymers.Cross-curricular standards emphasized in this labChemistry: Predicting sizes of molecules [HS-PS1-1], separating mixtures [HS-PS1-11], and understanding chemical interactions between substances [HS-PS1-3].Prior to the labInformation to cover in class: Basic introduction to chemistry for biologists (usually happens in the first chapter or two of the textbook). What are protons, electrons, and bonds?PhotosynthesisThe function of pigmentsCheck over the prelab:The TLC prelab focuses heavily on the chemistry involved in the experiment. This approach was intended to Help biology students develop a useful chemical foundation for other biochemical discussions (like micelle formation, protein structure, lipid structure, water contamination, etc.).Introduce students to concepts they will learn in future chemistry courses. The more students are exposed to this type of thinking, the easier it becomes. If you would like to focus on other important aspects of plant pigmentation, there are many good resources available.Material to prepare before the lab: TLC platesCut appropriately sized plates. Chromatography paper/silica plates work well. These are now so commonly used, you can get them from your regular chemical supplier or from Amazon.Provide other options for the solid phase. Paper chromatography uses many of the same components as TLC; allow kids to design parallel experiments on different surfaces like computer paper, water color paper, or filter paper. Three options total provides a good selection. If you provide more than one option, more groups will develop unique experimental designs.SolventObtain at least one good solvent like the chromatography solvent from Caroline Biological (Item number: Item # 746865, 9:1 petroleum ether and acetone)The American Chemical Society Guidelines for Chemical Laboratory Safety in Secondary Schools provide the following guidance on hydrocarbons and volatile organic compounds (like petroleum ether and acetone):These compounds are combustible or flammable and can irritate the skin. Used in a confined space, they can cause asphyxiation. They should always be used in a well-ventilated area or hood, and away from any open flames. Flammables and combustibles must never be heated on a hot plate.If you want to avoid using organic solvents, consider making this a paper chromatography lab. Set up mixtures of food coloring. You and your students can pretend it has been extracted from three generations of flowers. You can have students dot the dye on the paper and use water as a solvent. Have students identify the type of inheritance that you are modeling, calculate Rf values, and make some claims about hydrophilicity. If you use this approach, you will need to write a new point in the procedure to explain how to dot the dye on the paper. You will also need to establish a baseline for each dye and decide on the type of inheritance you are modeling. Additionally, you will need to update the questions for the lab report.If you want students to participate in the experimental design portion of the lab, obtain at least two other solvents. You can use any safe surplus solvent (ex. water, isopropyl alcohol). When students design their experiments, ask them which solvents they will be running. Ensure that they do at least one “good” run so that they can perform the pertinent calculations. NOTE: Water will cause silica to fall off TLC plates. If a group plans a water experiment, have them start with one plate before adding any additional trials.Three options total provides a good selection. If you provide more than one option, you will see more diversity in experimental design.GlovesCheck to see if you have the appropriate gloves for the solvents you will be using. You probably already have a chart in your desk, but if you do not, Berkeley has a convenient table for glove selection (). Developing chambers Beakers are the easiest to set up. They are also the easiest if you have limited glassware and a large class because you can run multiple plates in one chamber. Do not run more than three at one time; the solvents are quite volatile, so it is difficult to record the solvent front for every plate if the chamber is too crowded.Erlenmeyer flasks work well, but you can only run one plate at a time and you would have to prepare corks. Corks can be prepared by sticking a paperclip through them. Leave a hook for paper or plates.Test tubes are also reported as an option [author’s note: I have not tried this method, but it would be easy to scale up in a busy class, so it is worth mentioning]. If you are concerned about waste, or you want to run more experiments in parallel, consider this method. If you decide to use a test tube, you will need to cut the TLC plates such that the bottom has a triangular tip that touches the liquid phase. Make sure that students place the base line above the triangle piece. If the TLC plate is a little long, a cork can be used to hold it in place. If you use this approach, students should include approximately 5 mL of solvent.Pencils (at least one per group)Rulers (at least one per group)Pennies/other coins (at least one per group)Cameras (permission for cell phone camera use often helps speed up data collection, but you will need to discuss proper/improper use and ramifications of inappropriate use).Waste containersTrays (so that possible spills are contained)Extra paper toweling Recommended teacher pre-reading/watchingQuick (4:11) video on TLC: teacher pre-reading/watchingTLC written descriptions and labs for teachers on plant pigments structures obtained from ACS Guidelines for Chemical Laboratory Safety in Secondary Schools to personalize this lab for your classroom:Level of discussion for intermolecular interactionsTable 2. Does not introduce every intermolecular force. For students who struggle with abstract concepts, including information that will not be immediately applied may unnecessarily confound the lesson. However, if you are working with advanced students, you may want to provide a more in-depth overview. You may also want to have students predict certain interactions like dipole-induced dipole. Including this information in biology should help students build a more intuitive grasp of the biochemical topics in the course. If your students take chemistry before biology, you can update the prelab to an appropriate level of complexity. As written, it assumes students have only the minimal grasp of atoms provided in the first few weeks of a biology course. For example, if your students have an understanding of electronegativity, that would be a good addition to the discussion of nuclear strength. Concepts like Coulombic attraction could also be named more specifically. When teaching in a chemistry first setting, identifying, this lesson works well with electron configuration because you can discuss hydrogen’s 1s electron and why missing that electron creates a unique situation. Jargon was intentionally minimized so that students are slowly introduced to anic chemistry unit in your districtIf your district does not go over an introduction to organic molecules in the chemistry curriculum, consider updating the section on predicting intermolecular interactions to include molecules without simplified structures.Sample preparationIf you would prefer to use fewer TLC plates and reduce the spread of pigments, you can spend time preparing samples and dotting them onto your plates. However, the coin technique helps students get testing quicker and allows them to run their first plate while they plan the rest of their tests. It also is easier to set up, easier to clean up, and easier for struggling students to follow.If you have access to a spectrophotometerIf you have a spectrophotometer, you can have students cut out the bands of pigments. Place these isolated pigments in isopropyl alcohol overnight. The pigment should transfer to the alcohol. Test for absorbance at 360 nm. This method will allow for more in depth discussion of absorbance and connect with longer discussions of photosynthesis. If your biology class does not have time for this part of the experiment, consider sharing samples with a colleague who teaches chemistry; the process described fits in with several useful topics.Possible cross-curricular connectionsThe 2016 Massachusetts State Science and Technology/Engineering Standards for high school chemistry emphasize predicting sizes of molecules [HS-PS1-1], separating mixtures [HS-PS1-11], and understanding chemical interactions between substances [HS-PS1-3].It would be interesting to have a chemistry class report back to group members in biology. The information shared could go into either a joint lab report, or merely be integrated into the biological lab report. Some alternative cross-over chromatography options include the following:A larger plan unitStudents in Biology study different biological features of the plant. For example, they may analyze different independent variables such as growth in different conditions (light/dark, exposure to possible toxins, soil changes, seed location, temperature) and differing seed types. They may also analyze different dependent variables including viability, chloroplast activity, root growth. Plant samples can be provided to the chemistry class where thin layer chromatography can be used to separate different pigments. Figures, tables, and information can then be shared with the biologists for their lab reports. Most chemistry classes go over mixtures at the beginning of the year and intermolecular interactions midyear. Alternatively, the biology class can do the TLC and the chemistry class can help with more chemistry heavy analysis like determination of iron content in the plants (this requires many important chemistry skills). Discuss timing options with a chemistry teacher before the school year begins. The prelab is heavily focused on simple chemistry related to biology. Chemistry students could look at that material (or just the structure of the pigments) and write a brief paragraph to share with the biology students. They can make predictions about which pigments will travel farthest in different solvents and have the biology class record a brief description of where each pigment ended up on the TLC.Alternative purification labs can be utilizedNickle affinity resin selectively interacts with His-tagged proteins. If the biology class works with mutant organisms bearing his-tagged proteins, they could isolate the proteins from those organisms. After the biologists purify all cellular protein, chemistry students could perform the nickel affinity chromatography. The idea of interactions between proteins and the resin is powerfully demonstrated by this kind of purification. Conveniently, if science fair projects are popular at the school, a similar purification technique can be replicated for students doing biology projects. The His-tag is small and therefore less likely to interfere with protein functionality than common bulkier tags; if you need a tag, this one is a good option. Additionally, the nickel affinity resin can be recharged and reused (always good on school budgets).Some ways a biology class might integrate his-tagged proteins include:Transforming bacteria with a construct containing a His-tag. Bacterial transformation is already one of the AP Biology labs; this approach could utilize existing materials and standard operating procedures.Purchasing a genetically modified organism for behavioral tests, microscopy, or other lab work. Make sure the genotype includes a His-tag. One great source for mutant drosophila you can use as human disease models is the Bloomington Drosophila Stock Center (). ................
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