QUANTITATIVE ANALYSIS OF THE VITAMIN C CONTENT OF URINE SPECIMENS VIA IODOMETRIC TITRATION WITH AQUEOUS/ETHANOLIC IODINE

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1 Sciencemadness.org, Prepublications Section QUANTITATIVE ANALYSIS OF THE VITAMIN C CONTENT OF URINE SPECIMENS VIA IODOMETRIC TITRATION WITH AQUEOUS/ETHANOLIC IODINE SUBMITTED BY USER HEXAVALENT - AUGUST 2012 ABSTRACT Discussed in this paper is an assay of the vitamin C content of urine specimens determined by iodometric titration against aqueous/ethanolic iodine. It can be done as vitamin C is a reducing agent and iodine is an oxidant thus a redox reaction can take place, providing an ideal way by examining the volume of titre versus analyte to determine the content of this nutrient in urine, expressed in mg/l. INTRODUCTION Vitamin C, or L-Ascorbic acid, is an essential nutrient for animal organisms, and most mammals can produce and store it in sufficient quantities with the main excpetions of primates including homo sapiens and C. porcellus (household guinea pigs), who must consume it in their diets to avoid the disease scorbutus (scurvy). Unfortunately, humans also do not store excess Vitamin C provided by their diets indeed, very little of this nutrient is actually metabolised with most excreted unchanged. Approximately 3% of this is to be found in the faeces whilst the remainder is removed via urine along with other waste chemicals from the body. The concentration of vitamin C in urine can vary dramatically, from less than 10mg/L to several thousand mg/l depending on several factors, notably the amount of the nutrient actually consumed by the organism, the frequency and volume of urination, the time of day, the organism s state of health and more. Moreover, the typical concentration of ascorbic acid in fresh urine is between 100mg/L and 1000mg/L (1g/L). It has been known for a long time that L-Ascorbic acid is a medium-strength reducing agent[2,3,4] and iodine, being an electronegative halogen, is an oxidant. Thus, procedures known as iodometric titrations can be used in instances such as these to assay the concentration of the acid in substances, either to determine purity levels of reagents, in instances such as this and many others. The reaction that takes place between the iodine and the ascorbic acid (more specifically, the ascorbate anion) is somewhat complex but is shown to be that one mole of ascorbic acid reacts with one mole of iodine, producing two moles of iodide ions and one mole of dehydroascorbic acid. See figure 1 below for reaction. Figure 1: Reaction of L-Ascorbic acid with iodine [2,5] I 2 + 2I -

2 Thus, a suitable method for assaying the L-Ascorbic acid content of urine was determined, and involved preparing a standard solution of ascorbic acid in de-ionized water, noting the quantity required to fully react with a known volume of Gram s iodine solution (approximately, by mass, 3% KI & 2% free iodine in 70% EtOH), running the same reaction with the urine specimen and then calculating the ratio between the two to find the concentration of vitamin C in the urine specimen. Ordinarily, calculations would be suffice for this, however the Gram s iodine was originally prepared for staining microscope slides as part of a staining protocol. Thus, it was not prepared to a high level of accuracy, meaning that the method described above would yield more reliable and precise results. The former was also aided by repeating the urine titration to obtain a second result, from which the average would be calculated. The intention, therefore, would be to continually add the vitamin C sample (and then the urine on the second and third attempts) until the iodine was completely reduced to colourless iodide ions. Using the pure vitamin C, this would present no problem as the aqueous solution of it is colourless. However, as urine specimens can take on a broad range of colours, from very light yellow to very dark yellow, this presents a problem; when almost fully reduced, the iodine too is yellow, meaning it cannot be easily told when the iodine itself has been completely converted to iodide ions and when just the urine remains. Thus, to overcome this problem, an external indicator was employed; starch solution.[1] When the mixture became yellow as described above, 2-3 drops of starch solution would be added. Free iodine reacts immediately with starch* to produce a blue/black complex, and thus, by continuing to add the urine until a yellow solution (from the urine) is formed, it can be made sure that all the iodine has reacted. *Many of the details of the reaction are still unknown, but it is believed that the iodine becomes stuck in the coils of beta amylose molecules (beta amylose is a soluble starch, see Figure 2 for structure). The starch forces the iodine atoms into a linear arrangement in the central groove of the amylose coil. There is some transfer of charge between the starch and the iodine, cauing a change in the way electrons are confined, and so, a change in the spacing of the energy levels. The iodine/starch complex has energy level spacings that are just so for absorbing visible light; giving the complex its intense blue/black colour. Figure 2: Structure of β-amylose [5] HEALTH AND SAFETY The starch indicator in itself presents no significant hazard, but be careful of mold growing on old samples. Iodine is toxic, in the solid form emits toxic and irritating vapours and stains almost everything. [6] L-Ascorbic acid presents no significant hazard, but be careful of ingestion and inhalation of the powder. [6] Take care when using heat sources and any sharp objects used to cut and deliver samples e.g. scalpels and hypodermic needles for fluids to avoid cuts and burns. The urine sample may present a biological hazard be careful when selecting samples. Wear protective clothing, splash goggles and nitrile gloves at all times when carrying out procedures.

3 EXPERIMENTAL Note these procedures were carried out semi-microscale, meaning that counting drops of fluids often provided a more accurate and convenient determination of volume than attempting to use conventional measuring equipment, e.g. graduated cylinders and pipettes. Where used, all drops were supplied with a 10ml syringe fitted with a 21-gauge needle, and using online reference charts such as; (Copyright Sigma Aldrich 2012), the equivalent value in units such as millilitres can be determined. STAGE 1 PREPARATION OF STARCH SOLUTION INDICATOR A small potato was cut up and several small pieces, approximately 0.3cm 3 apiece, were added to a standard test tube containing 8ml of distilled wtaer. The tube was gently heated until the water boiled, and was kept at this temperature for 3 minutes, replenishing some of the water as necessary to keep the volume constant. After heating, the tube was allowed to cool and any suspended solids to settle out. The supernatant was then decanted into a small vial and kept in the refrigerator, where it may be stored for 2-3 days. STAGE 2 PREPARATION OF A STANDARD L-ASCORBIC ACID SOLUTION It was discussed previously that the average Vitamin C content in urine was between 100 and 1000mg/L. Thus, it was decided to prepare a standard solution in approximately the middle of these values, at 500mg/L or 50mg/100cm 3. Also, note that L-Ascorbic acid is prone to air-oxidation and is mildly photosensitive. Work carefully but quickly and store the final solution in an amber glass bottle or discard after use. 50mg ( moles) of pharmaceutical grade L-Ascorbic acid (purchased as pure, crystalline Vitamin C supplement from was carefully transferred to a 100ml beaker and the weigh boat rinsed carefully with 3x10m portions of de-ionized water into the beaker to ensure a quantitative transfer. 40ml of additional de-ionized water was then added slowly and the mixture stirred with a glass rod to completely dissolve the vitamin C. The solubility of L-Ascorbic acid at room temperature is far above the amount used here, thus the dissolving of the compound is fast and easy. The solution was then transferred carefully into a 100ml volumetric flask and the inside of the beaker and the glass rod rinsed with 2x10ml of de-ionized water. The flask was filled to the correct level and homogenized by shaking, before being transferred to an airtight amber-glass bottle. The headspace in the bottle was filled with dry butane from a small cylinder to purge out any remaning oxygen from the air. Parafilm was then placed over the mouth of the bottle and the cap screwed on tightly. Carbon dioxide, being cheaper and easier to obtain than butane at short notice, was not used due to its tendency to dissolve (albeit with equilibrium) into water, possibly changing the ph. STAGE 3 PREPARATION OF GRAM S IODINE As noted previously, the Gram s iodine was prepared beforehand for use as part of a staining protocol. It was made by dissolving 3% KI and 2% solid I 2 in a quantity of 70% EtOH, specifically 0.3g KI and 0.2g I 2 10ml of the solvent. Since its preparation, it has been stored in an amber glass dropper bottle and does not seem to have degraded in any way since its first preparation. One can also substitute just iodine in an alcoholic or aqueous medium for this solution. STAGE 4 DETERMINING A REFERENCE VALUE WITH THE STANDARD SOLUTION 2.00ml of distilled water, measured in a volumetric pipette, was added to a clean test tube and 10 drops of the Gram s iodine was added. The mixture was swirled and a small triangular-prism magnetic stir bar added. The tube was clamped above a magnetic stirring-hotplate with a sheet of white paper on the top surface to aid in seeing colour changes. Stirring was begun at a medium speed and the syringe was filled with the standard Vitamin C solution by poking the needle through the parafilm and inverting the bottle. Using a tally counter to aid in counting the number of drops dispensed, fully-formed drops of the Vitamin C

4 solution were allowed to fall into the test tube and mix with the iodine. When the solution became colourless once again, addition of Vitamin C was ceased and the number of drops dispensed recorded. STAGE 5 DETERMINATION OF THE CONCENTRATION OF ASCORBIC ACID IN URINE A small sample of urine taken from a person eating a regular diet (no excessively large or small amounts of Vitamin C- containing foods or beverages or supplements taken) was obtained and placed into an amber glass vial, again with butane filling the headspace and parafilm over the mouth. The procedure as above was replicated, but when the colour had just faded to a yellow colour, where small amounts of free iodine present would have been indistinguishable from the colour of the urine, 2ml of the starch solution was added, resulting in the instantaneous formation of a deep blue/black complex, showing that iodine was indeed still present. Vitamin C addition was continued dropwise until the dark colour faded and a yellow colour, caused purely by the colour of the urine, remained. Once again, the total number of drops used was recorded and the entire stage repeated once again to obtain a second value for the same urine specimen. RESULTS Here are the results obtained from the experiments detailed above, transcribed straight from the laboratory notebook; A Quantity of Standard Vitamin C Solution required to react with all Iodine 14 drops (±1 drop) B Quantity of Urine Specimen required to react with all Iodine Trial 1 9 drops (±1 drop) C Quantity of Urine Specimen required to react with all Iodine Trial 2 10 drops (±1 drop) D Average Quantity of Urine required to react with all Iodine ((B+C)/2) 9.5 drops (±1 drop) E Concentration of Vitamin C in Urine specimen ((D/A)*500) mg/L (7.14% potential error) Figure 3; Visual representation of the final result, showing the average range of mg/L concentration of Vitamin C in urine in red, 0mg/L and the mid-range point (500mg/L) in black and the value obtained in this experiment in blue.

5 DISCUSSION OF RESULTS As seen on the previous page, the concentration of Vitamin C in the urine sample was found to be mg/L, or g/L. This value is expected to be relatively accurate and reliable, as steps were taken to prevent both oxygen-caused and photodegradation of the Vitamin C, when in the form of the standard solution and when handling the urine specimen. Research has been done by John D. Kirschmann and noted that Vitamin C is primarily absorbed by the membranes of the mouth, stomach and upper part of the small intestine. He adds that the larger the dose, the less is absorbed, such as 80 percent absorption rate when you take under 250 mg, but only 50 percent absorption rate when taking above 250 mg up to 2 g*[7]. This could be supplemented by additional research into other compounds that are reported to help increase the efficiency of Ascorbic acid absorption, such as bioflavonoids [7]. The most realistic for this use would be non-ketone polyhydroxy polyphenol compounds which are more specifically termed flavanoids, flavan-3-ols (or catechins). Some researchers also note that taking the ascorbic acid in an esterified form can also help e.g. in compounds such as ascorbyl palminate, made using ascorbyl alcohol, or those where the ascorbate portion of the molecule came from the acid as in alkyl ascorbates. REFERENCES [1] The Illustrated Guide to Home Biology Experiments by Robert Bruce Thompson and Barbara Fritchman Thompson, First Edition, p O Reilly Media, Published 2012 [2] The Oxidation of Ascorbic Acid and its Reduction in Vitro and in Vivo by Henry Borsook, Horace W Ravenport, Cecil E P Jeffreys and Robert C Warner [3] Preparation of fine copper powder using ascorbic acid as reducing agent and its application in MLCC by Songping Wu (College of Chemistry, South China University of Technology) [4] Oxidation-Reduction Potential of Vitamin C by Henry Borsook and Geoffrey Keighley [5]Google Images and Wikipedia articles for structural diagrams [6] ScienceLab s MSDSs for Ascorbic Acid and Iodine [7] Screening for Vitamin C in the Urine: Is it Clinically Significant? by James A Jackson, Kelly Wong, Chad Kriver and Hugh D Riordan REAGENT SOURCING FOR THE AMATEUR L-Ascorbic acid Potassium Iodide Iodine Can be purchased as a vitamin supplement, either as a pure powder or as pills. If you buy pills, when dissolving the pill in water allow any undissolved solids to settle out before using the solution. These are likely to be binders and other insoluble contaminants. Can be purchased easily online. (Note not needed for this particular experiment, but part of proper Gram s iodine which is used as part of a staining process for microbiology experiments. Can be purchased, but much easier, convenient and legally safer for the amateur to prepare by the oxidation of potassium iodide solution (using hydrogen peroxide) acidified with hydrochloric acid. The peroxide can be purchased online or in pharmacies and drugstores, and the hydrochloric acid can be

6 found online or in stores as either Muriatic Acid for cleaning concrete or Spirits of Salt for other cleaning purposes. Starch solution As described above, prepared from boiling a small amount of potato (or any other carbohydrate-rich food such as rice or pasta). *See full article at