“Spectrophotometric Analysis Of Salicylate In Plant Tissues”
The Challenge:
After reading about salicylates in plants, formulate a testable hypothesis in which the measurable variable is change in salicylate production. Design an experiment to test the hypothesis.
Objectives:
- Use of a dichotomous key to identify plants
- To determine the amount of salicylate present in plant tissue using UV-visible absorption spectroscopy or colorimeter
- To compare various plant tissues with respect to the amount of salicylate present
- To design and carry out a scientific investigation
Science Concepts:
Plants produce a great variety of secondary compounds, many of which afford the plant a degree of protection from insects and other herbivores. Salicylic acid, a precursor of acetylsalicylic acid (aspirin), is produced by many species of Angiosperms. It is stored in large amounts in the leaves and bark of Willows (Salicaceae) and Birches (Betulaceae) and serves to protect these plants from insect attack. In Potato and Tobacco (Solanaceae), the amount of salicylic acid present in the plant is normally low, but increases to high levels in response to attack by pathogens. These high levels of salicylic acid trigger systemic-acquired resistance (SAR) in these plants, enabling the plant to fight off infections. Potato and Tobacco plants can be made to produce protective levels of salicylic acid by either simply scratching the leaves or spraying the plant with arachidonic acid or benzaldehydea sort of plant vaccination. Interestingly, a derivative of salicylic acid, methyl salicylate, is released into the air by virus-infected Tobacco plants. This vapor actually activates increased production of salicylic acid in neighboring uninfected plants, rendering them more resistant to viral attack.
The use of salicylic acid as an analgesic has a long history. More recently it has been shown to have anticoagulant properties, making it a useful drug for the treatment of some types of heart disease. It has been reported that strict vegetarians have markedly higher levels of salicylic acid in their blood than do non-vegetarians, even some of those who take a daily aspirin tablet. Some have suggested that the high levels of salicylic acid explain the lower rates of heart disease and some cancers among vegetarians compared to that of the general population.
The following products may contain salicylate compounds: Acne products, breath savers, bubble baths, cosmetics, fragrances and perfumes, gums – mint flavored, hair shampoos, conditioners, or sprays, herbal remedies, lipsticks, lotions, lozenges, mouth washes, muscle pain creams, razors with aloe strips, shaving creams, skin cleansers or exfoliants, sun-screens or tanning lotions, toothpastes, wart or callus removers.
However some people have sensitivity to salicylates. For this reason there is concern that agronomists trying to increase the levels of salicylic acid in plants may actually create plants that can be toxic to a small percentage of the population. Sensitivity to salicylate may produce some of the following symptoms: anaphylaxis (rare), asthma, breathing difficulties, changes in skin color, congestion, fatigue, headaches, hyperactivity, Itchy skin, rash, or hives, Itchy, watery, or swollen eyes, stomach aches or upsets, wheezing.
Review of Basic Concepts:
Chemistry students selecting plants for testing are reminded that the easiest ways to distinguish monocots from dicots is to study the veins in the leaf. Dicots have branched veins, while monocots typically have parallel leaf (although, there are exceptionsfor example, the opium poppy is a dicot, but its leaf resembles that of a monocot). More dicots have been found to contain alkaloids than monocots. Solutions, which are highly polar in nature, are soluble in polar solvents such as water but are not soluble in nonpolar solvents, or lipids. The converse is also true; most substances that are nonpolar will dissolve in nonpolar solvents but not polar solvents.
Procedure:
Collect the plant materials to be tested. Identify the plant and determine if it is a monocot or dicot. Dry the tissue*, grind, and mass 0.5 grams. Add 10 ml of 0.25 M NaOH and let stand 10 minutes. This process will extract the salicylate into the aqueous solution. While extracting the salicylate, prepare the standard curve.
* Fresh sample can be used. To use plants containing chlorophyll, prepare the blank from the stock plus Trinder’s Solution.
Standard curve preparation:
Materials: Spec 20, cuvettes (6), 100 ml of 0.01 M sodium salicylate, 10-ml volumetric flask, distilled water, Trinder’s Reagent, micropipettes.
1. Using a volumetric flask, mix the following salicylate standards, place in cuvette:
Blank: 7.5 ml Trinder’s, fill to volume with water.
1.50 mM: 7.5 ml Trinder’s, 1.50 ml of 0.01 M salicylate, water to volume.
1.25 mM: 7.5 ml Trinder’s, 1.25 ml of 0.01 M salicylate, water to volume.
1.00 mM: 7.5 ml Trinder’s, 1.00 ml of 0.01 M salicylate, water to volume.
0.75 mM: 7.5 ml Trinder’s, 0.75 ml of 0.01 M salicylate, water to volume.
0.50 mM: 7.5 ml Trinder’s, 0.50 ml of 0.01 M salicylate, water to volume.
2. Record the absorbance for each concentration with the Spec 20 set at 545 nm. Zero each time with the blank and start with 0.50 mM and work up to 1.5 mM.
3. Graph absorbances, determine the best fit line
Plant tissue analysis:
1. Pack a disposable pipette with a small amount of glass wool. Filter the solution.
2. Place 0.50 ml of filtered solution in flask, add 7.5 ml Trinder’s and water to make 10 ml of solution.
3. Record absorbance with Spec 20 or colorimeter, remembering to zero with a blank each time (see step 1 in standard curve procedure).
4. Calculate the amount of salicylate in the sample.
Teacher Notes:
You may use colorimeters instead of Spec20s. If you use a colorimeter, set the wavelength to GREEN.
1. The possible plant related studies using this basic approach are numerous. Students could sample a wide range of plants in order to simply see if salicylic acid is present. It is likely that there are some common species that have not been sampled. (eg. Dandelions). Another possibility would be to monitor the salicylic acid levels of a single plant throughout the growing season. Different tissues on the same plant could be examined, such as new bud, old leaves, etc. Comparisons between related taxa would be interesting, such as Thyme, Mint, Oregano, and Tarragon. With respect to health issues, the amount of salicylic acid in raw, boiled, baked, and fried sweet potatoes might be a nice study. Does cooking affect the levels of salicylic acid?
2. Standard curve data are supplied below for absorbances at 530 nm and 545 nm. The regression line derived from the 545 nm data appears to be slightly better when the formula for the regression line is: y = 0.51 x. To determine salicylate concentration from absorbances: salicylate conc. = absorbance / 0.51
3. The procedure with respect to plant tissue seems to be more robust. Measurable amounts of salicylate in 0.5 g of fresh tarragon have been obtained after just 5 minutes of reaction with 0.25 M NaOH.
Solutions:
Trinder’s Reagent:
0.40 g of Iron (III) nitrate 9-hydrate in 1.2-ml of 12 M HCl. Add water to 100 ml.
0.01 M sodium salicylate
0.16 g sodium salicylate. Add water to 100 ml
or&.0.14 g salicylic acid + 0.04 g NaOH. Add water to 100 ml.
