Vitamin C Assay in Fruit Drinks (Robert)

Title: Starch Assay for Relative Vitamin C Content of Drinks

Principle(s) Investigated: Titration of an aqueous chemical reaction with diagnostic endpoint color change is reproducible and allows quantitation of a reacting solution component.

Vitamin C [ascorbic acid] is a biologically important antioxidant compound.

Antioxidant activity prevents formation of the colored reaction product of starch and iodine.

Relative amount of Vitamin C present in a water-based drink may be determined if a known amount of added starch solution is first added and then titrated to its colored endpoint with an iodine solution. Relative Vitamin C concentration is seen by how much drop-wise additional iodine solution is first required in the solution to neutralize the antioxidant, before the starch-iodine color reaction occurs.

Standards: Matter and its Interactions:

MS-PS1-2: Analyze and interpret data on the properties of substances before and after the substances interact to determine if a chemical reaction occurs.

Crosscutting Concepts:

MS-LS1-3: Systems may interact with other systems; they may have sub-systems and be part of larger complex systems.

Science and Engineering Practices:

MS-LS1-1: Conduct an investigation to produce data to serve as the basis for evidence that meet the goals of an investigation.

Materials: Materials List (summary):

Safety Requirement (wet chemistry lab): Every student must use safety goggles during lab work. (Additionally, iodine solution stains skin and clothing, so lab coats and nitrile gloves should be used.)

Each student lab group needs its own:

Starch solution in a bottle, with dedicated disposable plastic dropper; a working solution is easily made using spray starch sold in pressurized cans for treating clothing for ironing. Spray a small amount into a glass of water until it becomes slightly cloudy; this is a high enough concentration of starch for the lab reactions.

Iodine/potassium iodide solution in a bottle, with dedicated disposable plastic dropper; schools typically buy this solution from chemical supply houses (biological supply houses such as Carolina Biological Supply also have it available), and it may also be purchased over the counter at any pharmacy.

The instructor needs a solution of Vitamin C, to demonstrate the effect of adding the antioxidant to an iodine/starch solution in which a dark blue endpoint color has already been reached. The instructor can then show the class that addition of Vitamin C removes the dark blue reaction color from the starch/iodine solution, and then, that adding more iodine to titrate the excess Vitamin C will again produce the dark blue starch/iodine reaction color in the solution. Vitamin C tablets are readily available in food markets and at pharmacies. Typical tablets each contain up to several hundred milligrams/tablet of ascorbic acid, which can dissolved in up to 50 mL of water for a working solution.

The teacher needs to pretest the various solutions and fruit drinks to determine that reasonable concentrations of the solutions have been made allowing reaching endpoints within 20 or 30 titration drops during the lab experiments. All the reagent solutions, above, are stable for weeks at room temperature, in clean bottles.

Clear glass or plastic cups to hold the endpoint reference solution, and for each or the 5 or 6 test drink solutions.

Solution volumes may be measured out using cups marked with fill lines, or in graduated cylinders. Transfer containers need to be rinsed out between solutions, to avoid cross contamination between test drinks, or reagents.

Additional comments related to Materials:

Various drinks, water-based fruit or vegetable drinks, and also milk, as well as teas containing fruit or vegetable extracts should be selected so that about 5 or 6 test drinks are available for the lab work. However, many commercial drinks contain artificial flavorings and minimal or no real fruit juice content, and many have arbitrarily added Vitamin C and other chemicals, some of which are also antioxidants. If the goal is to test relative Vitamin C content in authentic fruit or vegetable juices, it may be necessary to prepare the test drinks by using a blender on actual fruits and vegetables, to produce the test drinks.

It is necessary to have a starch solution to add in identical aliquots to each test drink, and also an iodine/potassium iodide solution. Each of these solutions needs to be given in separate containers to each group in the lab, for use on their test drinks. Each student group needs to prepare a standard reaction solution for visual endpoint reference, in another clear container identical to those that will hold each test drink.

Direct comparison of various water-based soft drinks for Vitamin C [ascorbic acid] content requires transparent glass or plastic containers to observe and compare a standard reaction endpoint and that found in each of the drinks in the experimental series. It is also efficient to use 20 mL aliquots of each test solution in the student lab groups, since a single commercial can or bottle of juice or milk or other soft drink typically contains about 400 mL of drink, which will then be sufficient for more than a dozen individual student lab groups during a lab class session. Limiting the test volumes to 20 mL/drink type also fixes the titration volumes to a reasonable range to quickly reach endpoints. Each group requires a bottle of starch solution; a bottle of iodine/iodide solution; a clear plastic or glass cup in which 20 mL of drink is easily held with additional room to swirl without spilling out for each test drink solution. Either a small graduated cylinder (50 or 100 mL) or a cup with a “fill to” line marked on it may be used to reproducibly transfer drinks from commercial container to a test cup, for each drink tested.

Accurate drop-wise addition of starch solution and also of the iodine/iodide solution used in the titration requires reproducible drop formation from a dropper to deliver a reproducible small volume of starch solution to each test solution, and also for accurate and reproducible drop-wise addition of iodine solution to each test solution. Typical titration volumes require a range from a single drop to as much as thirty drops, to reach the starch-iodine colored endpoints, and each drop added to a test solution should be easily formed and delivered from the dropper used. Bottles with screw-in glass droppers are frequently difficult to use, especially if the rubber squeeze bulb is old and has developed cracks in the rubber. Therefore, each group needs at least two (2) clear plastic disposable droppers, with one used for the starch solution delivery and the other to titrate the prepared test drink with the iodine solution.

Procedure: Before preparing the various solutions for the experiment, discuss with lab partners your predictions for the relative amounts of Vitamin C you expect to find in the test drinks. Rank the drink you expect to have the most Vitamin C as “number 1,” and then the other drinks as predicted “numbers 2, 3, and 4.” After testing the drinks, rank the drink which required the most drops of iodine solution to reach the blue/black endpoint color as “number 1,” and continue with the actual ranking of the other three drinks.

Record all data in the Data Table.

A) Make a reference solution

1. Label a plastic cup “reference solution.”

2. Put 20 mL of water into the cup.

3. Put 10 drops of starch solution into the cup.

4. Put 1 drop of iodine solution in the cup and swirl cup to mix.

5. Save this solution for visual reference purposes.

B) Test each of the drinks

1. Put 20 mL of “100% Lemon Juice” into a cup.

2. Put 10 drops of starch solution into the cup.

3. Add 1 drop of iodine solution and swirl cup to mix.

4. Continue adding iodine drops and swirling until you obtain a blue/black color similar to the “reference solution.”

5. Record the number of drops in the data table.

6. Repeat for the other drinks.

Data Table:

Student prior knowledge: Based on an earlier class lab in which various solid foods were wetted and exposed to iodine/iodide solution, students are familiar with the dark blue color produced in starch-containing foods, as diagnostic for the presence of “starch” in the foods.

Another earlier lab studying color change in pH indicators as acid or base was added to an aqueous solution gives students the understanding of “endpoint” and the quantitative use of “titration” with a reactant to produce a visible color change in a solution corresponding to a specific pH value.

Students need to understand qualitatively that Vitamin C and iodine react together before the iodine solution can react with starch, preventing the iodine-starch reaction from happening. The iodine-starch reaction does not occur until the Vitamin C is “used up” by additional iodine drops to the drink. Therefore, students can understand that “extra drops of iodine” corresponds with “amount of interfering Vitamin C,” and the drinks requiring more “extra iodine” drops have more Vitamin C in them.

These preliminary concepts, preceding the lab, are revisited the day after the lab by a Warm-up question at the start of the next class session: “In yesterday’s lab, __________________ reacted first with Vitamin C. When the Vitamin C ran out, iodine reacted with _______________ and turned a _______________ color.” [Answers: iodine; starch; dark blue]

Explanation: Elemental iodine (I₂) is not water soluble, so iodine solution contains added iodide ion (I^-) in the form of potassium iodide, to make the iodine into a solution in water. The iodine is present in the solution as alternating I₂- I^- species, which are linear in shape. Since the iodide ion shares its electron charge with neutral elemental iodine, this is called a “charge transfer” species. This form of iodine-iodide species is easily excited by light and electron energy transitions produce the soft brown/red iodine color in solution.

Starch produced in plants is in two forms of polymers, both made of glucose monomers linked end to end. The long linear starch polymer is amylose, and the other highly branched starch polymer is called amylopectin. Most of the starch (> 80%) is amylopectin, which does not react with iodine. But, the long linear amylose forms a helix in water, and the linear iodine-iodide species fits into the center of the amylose helix, where the amylose donates electrons easily to the iodine-iodide. This is also a charge transfer complex, with the amylose as the electron donor, and the resulting electron transitions in light produce the dark blue color of the iodine-starch reaction.

The dark blue starch-iodine color only comes from the interaction of iodine with linear amylose molecules, not with amylopectin. Therefore, the colored reaction only measures the amount of amylose present in the plant starch.

Vitamin C is produced in plants, but not in animals, and functions as an antioxidant to protect biological molecules in cells and tissues, since it oxidizes very easily, before other cellular species do so. Its reduced form is ascorbic acid, and its oxidized form is dehydroascorbic acid. Ascorbic acid reduces elemental iodine (I₂) to iodide ion (I^-), which destroys the alternating linear species (I₂- I^-) which produces the colored reaction with amylose. However, after addition of excess iodine-iodide solution to react with the ascorbic acid, and “use it up,” further iodine solution reconstitutes the (I₂- I^-), and the starch-iodine dark blue color is seen again.

Since the experiment begins with preliminary addition of 10 drops of starch solution, all of the test drinks contain starch to react with iodine solution. Those drinks which also contain Vitamin C in them will require dropwise addition of excess iodine solution to “use up” the Vitamin C first, after which they will turn dark blue in the iodine-starch reaction. A drink without Vitamin C turns dark blue immediately with the first drop of iodine solution. Drinks with Vitamin C in them will require additional iodine solution to turn dark blue, and we count the excess iodine drops to rank the drinks for how much Vitamin C they contain. The drink which requires the most excess iodine contains the most Vitamin C. This experiment ranks drinks by relative Vitamin C amount.

Questions & Answers: (1) The antioxidant qualities of vitamin C are present in artificial fruit-flavored teas and drinks, despite the partial or complete absence of actual fruit or vegetable juices in the drink. Artificial fruit-flavored drinks are made with more vitamin C than many natural fruit or vegetable drinks. Are artificial drinks with high concentrations of vitamin C better to drink than natural fruit drinks?

Vitamin C is found in rose hips and other easily cultivated and harvested plant sources, which makes it inexpensive to add to commercial soft drinks. It is a health benefit as an antioxidant, and is not produced naturally in our bodies. However, other added supplements commonly found in soft drinks are thought to be potentially unhealthy, including large amounts of simple sugars used to sweeten drinks, artificial flavorings to substitute for lack of a natural fruit taste, and chemical preservatives to retard spoilage. The potential benefit of high antioxidant protection from additional vitamin C can be overshadowed by problems from other additives in artificial soft drinks.

(2) Most commercial fruit drinks contain high fructose corn syrup as a sweetener, in addition to natural starch and sugar from fruit or vegetables. Consuming fructose and other added simple sugars can lead to diabetes. Why don’t food companies produce fruit drinks with natural starch and complex carbohydrate found in fruits and vegetables, instead of using high fructose corn syrup?

People have always enjoyed eating fruits and vegetables for their flavors, textures and naturally sweet taste. Many people today get most of their food calories from meats and meat products, and eat mostly commercially prepared foods, especially in cities. They eat a limited variety of fresh fruits and vegetables because it is cheaper to buy prepared foods, and it often does not require that the person eating it actually prepare and cook it. Agriculture in the United States today is largely done by large companies which find it most profitable to raise animals for meat on a limited variety of vegetables (corn, soy beans). Cattle today are mostly fed corn, and not grass or hay, which is their natural food. Large-scale corn production yields high fructose corn syrup, which is useful as a “value-added” component to almost all processed foods, including meat, itself. Many fruit-flavored drinks, including teas and sodas, are also made with high caffeine content, which, together with the intense sweetness of high fructose corn syrup, is addictive. It is a cheap way for food companies to sell profitable food products, and sustain profits. Many popular fruits and vegetables are grown in tropical environments, and not in the temperate region where American corporate farming exists. Fresh fruit and vegetables have short shelf lives, also, and much potential profit is lost to spoilage. Therefore, many food companies do not find it profitable to market natural fruit and vegetable drinks.

(3) Preliminary knowledge for students doing this experiment includes prior experience with acid-base titrations using color indicators such as bromophenol blue and universal indicator paper, and also with the starch-iodine reaction, and its diagnostic dark blue endpoint color. What is the purpose or benefit of their doing this similar additional lab experiment?

The reverse titration of vitamin C prior to reestablishing the visible dark blue starch-iodine endpoint is a multistep process. It requires a higher level of cognitive engagement than the simpler prior experiments, which were essentially single step.

There is a need for some attention to detail in the drop-wise addition of starch and iodine solutions, as well as for some clarifying small group discussion during the lab. Drinks which have suspended particulate matter, such as coconut milk or lemon juice, may have endpoints which are harder to define, or see clearly.

While the lab seeks to define relative amount of vitamin C present, it can also lead to discussion of the presence of iodine-reactive and nonreactive forms of starch, broadening student thinking about definitions, vocabulary and the complex real world they describe.

Applications to Everyday Life: With the presence of both starch and the iodine-iodide solution, we expect the familiar dark blue color of their complex to appear. However, only after back titration of vitamin C with iodine solution does that dark blue color arrive, determined by whether the antioxidant is present or chemically neutralized.

This suggests that the process we may consider with broader examples is “chemical regulation” in a biological context. The status of an independently present species determines whether potentially reactive partners in a biological reaction react, or fail to react. It is a good basis for broadening the class discussion.

In plants, hormones trigger developmental events. Many students, and even some teachers, fail to think beyond the simple model of “hormone present: transition is initiated.” Such transitions are also dependent on other markers of cellular status, such as accumulated metabolites, or level of water stress. Other biological events, such as passing a cell cycle checkpoint, or perhaps, forming a memory, are likely dependent on presence or absence at a critical moment of an otherwise independent factor, outside the direct process we may study and consider.

The drop-wise approach and defined arrival at a visible endpoint in this assay’s titration is useful for reinforcing habits of observation and patience in experimental technique, and leads naturally to discussing systematic bias in measurement, and whether the errors are significant to results, or not. There is only a requirement here for determining the relative amount of vitamin C in a series of fruit drinks, and not the absolute concentration. A small systematic overshoot of endpoint is not significant for our focus on relative amount across drinks. It could be a very significant error in absolute quantitation of concentrations. The discussion would focus on the goal of an experiment as determinative in what we require in our practice and methods of doing the experiment. What is "good enough"?

The starch-iodine reaction's dependence on quantity of ascorbic acid [vitamin C] in the sample allows another lab exercise: The quantity of ascorbic acid changes over time in a fruit or vegetable, as it ripens. It would be interesting to test fruits or vegetables recently purchased, to see how long a vender presents them for sale.

Finally, here is an opportunity to discuss with students why starch-containing foods, such as cooked rice, or baked bread, change their texture and hardness as they become “stale.” Students may think it is mainly due to loss of water content (moisture), while it is mostly due to selective crystallization of amylose into hard crystals separate from the surrounding amylopectin in the starch. This is why some of the dry, hardened texture of “stale” bread and stored, cooked rice is reversed by reheating the bread or rice. The amylose crystals melt and mix again with the surrounding amylopectin in the food, and it softens and tastes better again. Relative amounts of amylose and amylopectin also explain the differences in taste and texture of different varieties of potato, or of cereal grains. The discussion could provide context for the effects of assuming a model of a process, versus examining experimental data and what is known about a system, to try to understand how seemingly independent parameters interact in a complex system.

[This lab exercise was developed by Mr. Kevin Takeuchi, my cooperating mentor teacher at Sutter Middle School, Winnetka, CA, where I have been a student teacher under his guidance, in 8th grade Physical Science class, Spring semester, 2017. The extended discussions, above, are mine. I wish to acknowledge my gratitude to Mr. Takeuchi for his guidance and mentorship.]

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