Laboratory

Last semester in class, in lab, and in office hours, you asked great questions about molecular structure and chemical reactions. These questions, which embody curiosity, wonder and (occasionally) confusion, are drivers of scientific research. For science to shed light on your questions, they must be connected with detailed, quantifiable results. Learning how to frame your questions in a scientific way is one of the goals of this project and something to be mindful of as your write your proposal.

Another goal of this investigation is to further your experience with the literature, techniques and instrumentation of the organic chemistry laboratory. The options that are open to you contain within them many standard protocols of lab work. Keep this goal in mind as you prepare your proposal, so that you include a variety of activities.

During the first week of the investigations, we will meet for brainstorming. You can use the experiments that follow as a starting point. By the end of this time, you will submit a proposal with your group. Over the next week, I will gather the materials that you need to begin your research and make procedures available for that work. By week 3, please complete the final draft of your proposal. Make sure to keep a detailed lab notebook as you progress. Every other week, your group will hand in a brief progress report. Finally, you will hand in a final lab write-up and give presentations during our lab time in the last week of the semester.

LAB SYLLABUS

Faculty contact information

Introduction

Lab this semester will be composed of a single project that you will propose and then carry out. Your research will closely mimic an independent research project. It will be connected to natural product chemistry and will include some biochemistry techniques as well as organic techniques.

There are several rationales for this extended lab. First, it will give you first hand experience in thinking through a scientific process yourself, a natural extension of some of the design-based labs that you did last semester. Second, your writing and presentation skills will be focused on as you discuss and present your own research. Third, you will learn common organic and biochemical lab techniques that are commonly used in many research labs, thus providing you will essential skills for future employment. Finally, because many students in organic chemistry are biology majors, this lab will help to connect the disciplines.

Attendance and participation in lab are mandatory and count toward your grade (see grade distribution below). Additionally, there may be some instruments, such as spectroscopic instruments, that you will need to use outside of lab hours due to limited availability.

During each lab meeting, you and your partner will meet with me for the purpose of identifying where you are where you are going in your research that week.

Assignments

There will be several different types of assignments. All of the assignments will be completed with your partner(s). First, you will write a research proposal, describing the question that you are going to answer and the experiments that you will do to answer that question. Second, you will write a progress report every other week. Third, you will write a final report. Finally, you will present your research. Details and expectations for these assignments will be discussed in lab.

Special needs and special arrangements (such as making up labs)

If you have any special needs such as a documented learning disability or other situation that limits your access or ability to participate in class or lab, please discuss the situation with me as soon as possible so we can make appropriate arrangements. Assistance with accommodations can also be found at the Office of Student Life, room 111 Reid Centennial Hall.

If you must miss a lab, please give me at least 48 hours notice so we can work out alternate arrangements. Of course, circumstances beyond all of our control happen. Don’t panic, but make sure to speak with me as soon as possible. There will be no incompletes awarded for students with less than an 80% average at the time of the incomplete. The course cannot be dropped after March 26. Plagiarism will be dealt with in accordance with college policy.

Summary of Grading

93-100%	A	83-86%	B	73-76%	C	60-66%	D
90-92%	A-	80-82%	B-	70-72%	C-	<60%	F
87-89%	B+	77-79%	C+	67-69%	D+

Note that each lab will begin with directions on how to use instruments and carry out other techniques, as specified below, and then you will work on your own project.

I. Some starting points for plant extractions. These suggestions are adapeted from Raphael Ikan “Natural Products: A Laboratory Guide” Academic Press, 1991 and Kaufman et al. “Natural Products from Plants” CRC Press, 1998. Underlined terms are described in Brielmann’s “Phytochemicals: the chemical components of plants”.

Obviously, all of these experiments are abbreviated here. I will provide you with the full procedures for whatever experiments you choose. As you design your investigation, you may need to develop your own procedures, consult the literature for procedures that will better serve your needs, or apply these experiments to new situations.

A. Terpenes

1. Isolation of capsanthin from paprika. Capsanthin is a carotenoid that can be extracted from paprika pods. It is a red compound, but turns blue upon acid treatment. You could characterize the protonated and deprotonated forms of the molecule and model the effects of the two on visible absorbance. You could compare the structure and biosynthesis of the red/blue moiety to the anthocyanins. You could measure its antimicrobial and antioxidant activity.

Capsanthin

2. Carotenoids in fruits and vegetables. Carotenoids occur in plants and provide color in the yellow to red range for many fruits and vegetables. Beta-carotene, in particular, is reported to have anti-oxidant activity, although it is likely that all of the carotenoids do because of their extensive conjugated bond network. You could isolate and characterize a variety of carotenoids from fruits and vegetables such as carrots, tomatoes and oranges. You could compare their antimicrobial and antioxidant activity.

3. Isolation of stigmasterol from a variety of sources. Soybeans, soybean oil, coconut oil, potatoes and many other plants contain stigmasterol. The extraction process is interesting, involving functionalization to afford pure product. The historical characterization (pre-NMR) is also interesting, involving ozonization, hydrolysis and reduction. It might be interesting to examine how natural products were identified in the 1930s and compare that to today.

Stigmasterol

4. Converting citrus to spearmint. All citrus peel oils have D-limonene as their major constituent. In a three-step synthesis, L-carvone, which is oil of spearmint, can be enantioselectively prepared. You could compare the structure of the product to a standard sample of oil of spearmint, and also to the enantiomer, oil of caraway. You could compare the antimicrobial and antioxidant activity of each. You could compare yields of D-limonene from a variety of citrus oils.

5. Converting a-pinene to camphor. a-Pinene is found in the oils of conifers and turpentine oil. It can be converted to camphor via a stereospecific rearrangement. Camphor is used as an antiseptic, among other applications.

6. Removing the bitter component of citrus juices. Cyclodextrins are basket-shaped molecules with hydrophobic interiors and hydrophilic exteriors. Thus, hydrophobic molecules in aqueous medium will preferentially complex with the interior of the cyclodextrin. If the cyclodrextin is supported on a solid-phase polymer, then the hydrophobic bitter components (limonin, nomilin and naringin) can be easily removed from the citrus juice. Typically, cyclodextrin complexes can be de-complexed by heating them slightly, and thus you could recover the bitter components. It may be interesting to try this technique with other hydrophobic molecules, as well as compare concentration of bitter components in several different juices.

B. Phenols

1. Isolation of hesperidin from orange peel. Hesperidin is a nonbitter flavenoid that is the predominant flavenoid in lemons and oranges. It is reported to decrease the fragility of blood capillaries. It can be extracted from the dry peel with petroleum ether and methanol or by alkaline extraction of chopped orange peel.

Hesperidin

2. Isolation of rhein from rhubarb root. Rhein is a quinone, one of a large group of pigments ranging in color across the full visible spectrum. Quinones play a part in the oxidation-reduction processes of living matter (thus they are often antioxidants) and some are antibiotics. Rhein can be isolated from aqueous extracts of rhubarb with methyl isobutyl ketone. You could isolate several of these phenols (hesperidin, rhein, anthocyanins, etc) and compare their antimicrobial and antioxidant activity.

Rhein

3. Identification of phenols and quinones from defensive secretions of beetles. Blaps, flour beetles or any other tenebrionids secrete a strong lachrymator (a tear-inducing compound) when disturbed. You can collect this secretion in a capillary tube and analyze it with GC/MS or other instrumentation. And, yes, I am aware that beetles are not plants.

4. Color changes of anthocyanins at various pH values. Anthocyanins range in color across the visible spectrum, possibly dependent on their number of hydroxyl substituents. Changing the pH drastically changes the color of these compounds in solution. The pH of sap, however, is typically 5.5 and so how color is controlled in plants is still a matter for debate. It has been proposed that tannins and other compounds, both metals and organic, could complex to anthocyanins to act in co-pigmentation. Anthocyanins occur in a wide variety of plants including roses and in red cabbage. You could compare their antimicrobial and antioxidant activity. You could also investigate the color of anthocyanin complexes with metals and organic compounds as a function of pH to see if that is how color is controlled in plants.

General structure of several anthocyanins. The R groups are either –H or –OH.

C. Tetrapyrroles: Chlorophyll extraction and use in catalysis. Chlorophyll is a light harvesting compound that contains a porphyrin moiety complexed to a magnesium(II) ion. Porphyrins are widely used in catalysis as well as electron transfer applications (potentially in solar panels, for example). It might be interesting to see if you could use the chlorophyll as a catalyst in oxidation reactions, such as the oxidation of lignans in paper pulp bleaching (see carbohydrates below). You would probably need to replace the metal, first, but maybe not. You could also synthesize porphyrins or extract them from petroleum crude.

D. Carbohydrates: Isolation of wood cellulose and catalysis. Delignification of wood (digestion and bleaching) is typically performed by treating wood with sodium hypochlorite, NaOCl. In a move to reduce chlorine releases, some paper companies (including Potlatch) are moving toward destroying the lignin with hydrogen peroxide and a metal catalyst, among other techniques. You could synthesize and compare the effectiveness of several different catalysts.

E. Proteins: Isolation and crystallization of lysozyme from albumen. Lysozyme is a protein with a molecule weight of about 17500 kDa (small for a protein). It lyses the cells of various bacteria and is considered an antibiotic because of this action. It can be isolated from egg albumen and crystallized in a variety of different ways. You could send the crystallized protein off to University of Idaho for characterization or you could see if the crystallized protein still catalyzes its lysing reaction.

F. Alkaloids: Isolation of piperine from black pepper. Not that you will be eating this, but piperine is tasteless as first and then sort of creeps up on you with a burning sensation. It may be initially tasteless because of its low water solubility. It can be straightforwardly extracted from black pepper with ethanol. This would be another interesting molecule to investigate its antimicrobial and antioxidant activity, as well as looking at solubility. Maybe you could functionalize piperine to make it more water soluble, but with the same action.

1. Common plants that have been reported to have interesting medicinal properties. Green tea, garlic, lavender, ginger are all easy to come by and have been widely reported have antioxidant and/or antibacterial properties. You can extract various components of these to look for the bioactive compounds.

2. Natural plant dyes. Lichen, crocus flowers, black walnuts and indigo are just a few examples of the plants that provide us with dyes. You could look at the history of natural dyes and investigate various mordants. You could also explore the role of various components of the dyes (i.e. which compounds are actually the dyes and what structural characteristics do they share?)

II. Identification and characterization techniques

A. Acid hydrolysis. Some compounds are composed of several components such as sugars and aromatic compounds that are separable by treatment with acid or base. Separating the components often simplifies identification.

B. Color reactions. The color of some highly conjugated compounds depends on pH. Identifying the color change with protonation state offers some structural evidence. It is also really cool to watch.

C. UV/vis spectroscopy. Quinones, carotenoids and other conjugated materials absorb in the UV and visible range. This can sometimes be used for identification and always for characterization.

D. IR spectroscopy. Compounds can be identified when compared to a standard. In general, IR is commonly used to establish functional groups.

E. Gas chromatography (GC). Mixtures of volatile compounds can be separated and each component’s retention time on the column can be measured for identification purposes. A standard sample is required for comparison.

F. Gas chromatography/mass spectrometry (GC/MS). Mixtures of volatile compounds are separated and then the molecular mass of each component is measured. A standard sample is not required but is helpful.

III. Biochemical assays

A. Antimicrobial activity. Bioassays will be conducted using the Kirby-Bauer method for antimicrobial susceptibility. This disk-diffusion assay employs a sterile filter disk that has been impregnated with the crude plant extract. The solvent is allowed to evaporate leaving behind the non-volatile compounds. The disk is placed on a microbial lawn and the zone of inhibition around the disk is measured as the antimicrobial activity of the extract. For each extract, three dilutions will be assayed (undiluted, 1:10 dilution, 1:100 dilution). The extracts will be assayed for their putative antimicrobial properties against a broad range of representative microorganisms including Escherichia coli (a gram negative enteric bacterium), Pseudomonas aeruginosa (a gram negative aerobic bacterium and commonly antibiotic resistant pathogen), Staphylococcus aureus (a gram positive bacterium), and Candida albicans (a human pathogenic fungus that has become a model organism in fungal pathogenesis).

B. Nicking of plasmid DNA. Plasmid DNA is a circular piece of DNA, typically found in bacterial cells. It is part of a convenient and sensitive assay for oxidative damage to DNA. In this assay, supercoiled plasmid DNA (Type I) is isolated from E. coli and is incubated under the desired conditions. When one strand of double-stranded supercoiled plasmid DNA is oxidatively damaged, one of the strands breaks, leading to an open circular form of plasmid DNA (Type II). When both strands of double-stranded supercoiled plasmid DNA are oxidatively damaged, both of the strands break, leading to linear DNA (Type III). The three different types of DNA can be separated by electrophoresis through an agarose gel and how much of the DNA is damaged can be visualized. You could compare the oxidative damage done to plasmid DNA by a variety of the natural products that you isolate above, or you could compare their antioxidant activity. In other words, you could add a known concentration of your natural product to the plasmid DNA and then expose the mixture to hydroxyl radical, which cause oxidative DNA damage. You can then determine if your natural product prevents DNA damage, and is therefore an antioxidant.

C. Catalase activity. The chemical reactions involved in keeping living organisms alive are almost always catalyzed by enzymes. These are large (around 10,000 to 30,000 g/mole), highly organized molecules that catalyze a specific chemical reaction. Catalase is a common enzyme responsible for catalyzing the transformation of hydrogen peroxide (H₂O₂) into oxygen and water. Hydrogen peroxide, besides being an over-the-counter disinfectant, is produced by another enzyme, superoxide dismutase, in an attempt to dismutate superoxide (O₂^.-), which is a byproduct of respiration. In other words, we need oxygen for respiration, but it’s a very reactive molecule particularly when it has an extra electron, and, even in our carefully controlled respiratory process, some of the superoxide molecules escape. Unchecked, they could damage all sorts of important molecules in our cells, like DNA and lipids. So, we have a series of enzymes, superoxide dismutase and then catalase, to turn the superoxide molecules into less toxic molecules.

You can easily observe catalase activity by observing foam formation on potato pulp after the addition of hydrogen peroxide. You can measure the rate of activity of catalase at several temperatures and measure the relative catalase concentration of different food products like fruits, vegetables and meat. You may also want to delve into the structure of catalases, and learn how to use Rasmol and the Brookhaven Protein Data Bank to visualize the enzyme. Finally, there are many ways to mimic catalase activity using metals without all of the amino acids attached. You could explore how our body protects itself by using enzymes rather than isolated metals by looking at the damage to lipids (like vegetable oils) in the presence of hydrogen peroxide and metals and comparing that to the damage to vegetable oils in the presence of potatoes. You could explore the inhibition of catalase with a variety of natural products as described above and come to some conclusions about the mechanisms of inhibition of catalase.

Comparison of antioxidant activity of polyphenols from green tea and vitamin C from oranges.

I have read that green tea and vitamin C are both antioxidants and I am curious about which is the better antioxidant. One of the purposes of this lab is to practice turning a subject that I am curious about into a scientific investigation, and the first step in doing that is to ask a question that can be answered with experiments.

The question that I am curious about, that will drive me to do my research is “Which is the better antioxidant, orange juice or green tea?”. While this is certainly a valid question and is important, it is not a scientific, or testable question. There are several reasons that it is not testable. First, the word “better” is nonspecific and nonquantifiable. In the lab, there is no test that tells me if something is better than something else. “Better”, for better of for worse, is a subjective conclusion that you can draw, but does not lead naturally to a laboratory experiment, so I must be more specific in my wording. Second, orange juice and green tea are not actually the antioxidants that I am interested in, they are mixtures of many different molecules, and, as a chemist, I need to be specific about which molecules I am going to test.

To modify my question, to make it into a scientific question, I first define what I mean as “better”. Here, the compound that protects DNA from damage by hydroxyl radical at the lowest concentration is the better antioxidant. Then, I identify my active molecules, they are polyphenols and vitamin C. My question then becomes “which protects DNA from damage by hydroxyl radical at a lower concentration, vitamin C from orange juice or polyphenols from green tea?”.

Notice that I had to know a little bit about the definition of antioxidants, and about the active components of the drinks, to modify my original question and make it a scientific question. Do not be daunted! Your text is a good resource, as is the background on natural products. Also, you can ask me and if I don’t know, I’ll help you with the literature search (something you will need to do for your project, anyway).

Now, before I can get to testing “which protects DNA from damage by hydroxyl radical at a lower concentration, vitamin C from orange juice or polyphenols from green tea?”, there are some other questions that I need to answer. First, I will discover “what is the concentration of polyphenol in an average cup of green tea?” and “what is the concentration of vitamin C in an average cup of orange juice?”. These are the sorts of questions that I could look up the answer to, but I will need to isolate the components anyway, so I might as well do the extractions to get my numbers. Once I had done the extractions, I will ask “Is what I think is polyphenol, really polyphenol?” and “Is what I think is vitamin C, really vitamin C?”. To answer these questions, I will do some spectroscopy and compare my samples to standards.

After I was satisfied that the compounds were pure and what I wanted them to be, I will then ask my final question “which protects DNA from damage by hydroxyl radical at a lower concentration, vitamin C from orange juice or polyphenols from green tea?”

When writing my final report, I will conclude by discussing the question that drove my research in the first place, “Which is the better antioxidant, orange juice or green tea?”. I will describe what I mean by better, and compare the activity of orange juice and green tea. I will also need to describe how my research fits in with the work done by other scientists in the field.

Week	lab activity	assignment due at the beginning of Lab
1	Introduction to the lab and group discussion followed by breaking into teams for initial assembly of project	Rough draft of proposal (Due at the end of lab)
2	Discussion of each group’s plans	Proposal: advanced draft (i.e. not a first or second draft)
3	ChemDraw	Proposal: final draft
4	Review last semester’s techniques
5	Review last semester’s techniques	Progress report
6	The primary literature of organic chemistry
7	UV/vis spectroscopy	Progress report
8	Gas chromatography (GC)
9	Gas chromatography/mass spectrometry (GC/MS)	Progress report
10	Bioassay for oxidative DNA damage and protection
11	-	Progress report
12	-	Final report: early draft
13	-	Final report: advanced draft
14	-
15	-	Final report: final draft
16	Check-out and clean-up

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