Environmental Health: Science, Policy and Social Justice Fall quarter 2008 Lab 3 Partition coefficient (n-octanol/water), P: shake

flask method (TSCA Sec. 799.6755) EPA has a guideline for this method. To find this guideline online follow these links: - Start from www.epa.gov/oppts, - from the left menu click on Test Methods and Guidelines, - select OPPTS Harmonized Test Guidelines and - select section 830 Product Properties Test Guidelines - select the method 830.7550 and save it as pdf file Definition Partition coefficient (P1) is defined as the ratio of the equilibrium concentrations (Ci) of a dissolved substance in a two-phase system consisting of two largely immiscible solvents. The P therefore is the ratio of two concentrations and is usually given in the form of its logarithm to base 10 (log P). In this case for n-octanol and water
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and C log P = log oc tan ol Cwater The partition coefficient is a very critical property that helps describe the relative water solubility and lipophilicity of a compound and therefore the properties of environmental fate, bioaccumulation and biological distribution. See posted USGS publication regarding the importance and difficulty in estimating logP for DDT and DDE (website). However, logP is valid only for non-ionizable compounds. The water solubility for ionizable compounds varies with pH and the logP value is not adequate to describe their distribution between oil (octanol) and water. In this case the distribution coefficient (D) is used instead, and the logD respectively is: C ol log Dow = log ionized oc tan nonionized Cwater + Cwater D and logD take into account the pKa of the compound and the pH of the aqueous
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Note: it is also common to see the partition coefficient indicated with Kow and logK instead. The P is preferable because there are many constants that use the K letter.

solution: 1 log Dacids = log P + log pH - pKa ) (1+ 10 and 1 log Dbases = log P + log pKa- pH ) (1+ 10 In this lab you will use a test substance, Phloxine B a red dye used in cosmetics that is relatively safe (no toxic substances yet!), for the purpose of applying the classic and commonly used shake flask method to determine the distribution coefficient, D and logD. The test substance was also chosen because it is ionizable and it will allow us to determine whether a substance like it could be absorbed in the gastrointestinal tract, how the pH in the GI tract would determine the absorption site/level (e.g. stomach or intestine) and how the substance would behave once it is in the circulation. Description of the test procedure Preparation of the solvents n-Octanol: Analytical grade n-octanol Water: Distilled or twice-distilled water Phosphate buffer at pH 7.4 Acetate buffer at pH 4.0 Presaturation of the solvents this has been done for you by lab staff! Before a D is determined, the phases of the solvent system are mutually saturated by shaking at the temperature of the experiment. For doing this, it is practical to shake two large stock bottles of purified n-octanol or distilled water each with a sufficient quantity of the other solvent for 24 hours on a mechanical shaker, and then to let them stand long enough to allow the phases to separate and to achieve a saturation state. Thanks Jenna! Preparation for the test For the purpose of this test the substance of interest is shaken in a closed container filled with the two solvents. The two solvents are then separated and the concentration of the substance in each phase is determined with an analytical method. Use separatory funnels, 125ml volume. The minimum quantity of Phloxine B required for the analytical procedure is determined by the detection limit of the method (for absorbance the minimum concentration detected is 0.5ppm = 0.5mg/L) 1.25mg/L (or 1.25g/ml)

Test substance The test substance for this type of determination should be the purest available. A stock solution of Phloxine B is prepared in n-octanol with a mass concentration of 300 milligram/liter (mg/L) [=300 micrograms/milliliter (g/ml), or 0.3mg/ml], stored under stable conditions (stable up to 5% w/v, at room temperature for 7days). The compound contains two ionizable groups and is shown here as the disodium salt. However, only one pKa is reported, presumably referring to the carboxyl group. Q* Give the equivalent mole/L concentration for Phloxine B stock solution Name MW CAS EINECS Colour Index Reported logPs: Solubility in water pKa Test conditions The test temperature should be kept constant ( 1oC) and may lie in the range of 20o25oC. Performance of the test 1. Establishment of the partition equilibrium Read the instructions carefully before you start working, to avoid mistakes. If in doubt ask the instructor or lab aid for help. Label 2, 125ml-separatory funnels A, B. Sep. funnel A: Use aqueous phase at pH = 7.4 (phosphate buffer) Sep. funnel B: Use aqueous phase at pH = 4.0 (acetate buffer) Calculate the appropriate volume of Phloxine B stock solution you need to add into 5.00ml n-octanol such that the final concentration of Phloxine B in 5.00ml noctanol is 15mg/L (15g/ml). 30mg/L (30g/ml) Phloxine B 829.66 g/mol 18472-87-2 242-355-6 CI 45410 9.99 (for the acid form) 2, 2.02 (for ionized form)[1] >10% (w/v) (pH 8.5) 4.29 (acid)

European Scientific Committee on Cosmetics and Non-Food Products (SCCNFP) pdf on program website (downloaded from: http://ec.europa.eu/health/index_en.htm).

Q* What is the volume of Phloxine B stock you need to add to each funnel? What is the dilution you are making? The total accurately measured amounts of the two solvents in each funnel are 5.00ml n-octanol and 100ml 25ml aqueous buffer. ***make sure the TOTAL n-octanol volume in the funnel is 5.00ml including the added Phloxine B stock!! Q* What is the volume of n-octanol that you should add to the funnel before you add the Phloxine B stock? Add the appropriate volume of Phloxine B stock that you calculated above to each funnel. Insert the glass stopper and carefully invert and vent until no more gas is released and shake the funnel 100 times by inverting it 180o rapidly as demonstrated by the instructor (perform 100 rotations in 5 minutes). Make sure that you release the pressure frequently using the stopcock. Let the funnel stand for a few minutes at room temperature to allow the layers to settle and remove the glass plug. Handle the funnel very carefully following separation to prevent any mixing of the separated phases.

2. Sampling from each phase For the determination of the D, it is necessary to analyze the concentrations of the test substance in both phases, n-octanol (o) and water (w). Take extra care to avoid mixing the two layers and contaminating one with traces of the other. It is better to leave some material behind than to accidentally disturb the layers and contaminate them. Label two clean, dry Erlenmeyer flasks or beakers Aw, Bw to collect the aqueous phases (w), and label two clean, dry scintillation vials Ao, Bo, to collect the noctanol phase (o) from each funnel (A, B). Carefully draw off the lower aqueous layer into a properly labeled, clean, dry Erlenmeyer flask or beaker. Cover with parafilm. Discard a few drops between the two layers into a labeled waste container. Draw the upper organic layer into another properly labeled clean, dry scintillation vial and cap. These two containers now contain your samples of unknown 4

concentration. You will later take aliquots of each of the two phases to use in the analysis. 3. Analysis In the next step you will determine the concentration of the dye in the two-separated phases. There are several choices of analytical methods, depending on the type of chemical and available instrumentation: (A) Photometric methods. (B) Gas chromatography. (C) HPLC. (D) Back-extraction of the aqueous phase and subsequent gas chromatography. For the purposes of this lab we will adopt a colorimetric method and use a spectrophotometer. In order to determine the concentration of Phloxine B in the unknowns collected above you need to prepare a standard curve (see Lab 1). Standard curve For preparation of a calibration plot (standard curve) for Phloxine B determination, follow these steps: 1. Label five separate test tubes: S1, S2, S5 for Standard 1, 2, etc for each solvent (aqueous phosphate, aqueous acetate and octanol) and one tube for Blank, labeled Bo Bw for each solvent. 2. Prepare the dilutions of Phloxine B (standards) for final concentrations of Phloxine B: 1, 3, 5, 10, 15mg/L in 6ml 4ml final volume. Use the aqueous stock of Phloxine for aqueous standards dilutions. Q* Prepare your calculations for the dilutions ahead of time and submit with lab report! a. Add the pre-calculated volume of solvent in each tube b. Add the appropriate volume of the Phloxine B stock solution (300mg/L) to each tube, so that you achieve concentrations in the range of 1-15mg/L in 6ml 4ml final volume. 3. Measure the absorbance of the resulting standard solutions at 538nm for the aqueous phases and 550nm for the n-octanol phase using the appropriate blank. Note: assume that the absorbance reflects both the ionized and the non-ionized

forms of Phloxin B. Determination of Phloxine B concentration in unknowns 1. Measure the absorbance of the separated phases (unknown concentrations) at 538nm or 550nm for water and n-octanol phases respectively, using the appropriate blank as a reference. 2. If any of the readings from the spec are outside the range of your standard curve prepare an additional dilution so that your reading is within range. Clean Up: Dispose water solutions down the drain in the hood, and collect n-octanol solutions into labeled waste container in hood. Wash all glassware with soap and water. Return separatory funnels and other equipment to the appropriate storage location. Clean up all spills on benches and inside fume hoods. Data and calculations Prepare a plot of each standard curve. Calculate the unknown concentrations of Phloxine B in each phase (n-octanol, water) for each test (A, B), using the appropriate standard curve and mark them on the standard curve. If you made any dilutions, calculate the unknown concentrations of Phloxine B using the formula C1V1=C2V2 (show your calculations) Calculate the amount of Phloxine B in each phase (show your calculations) Calculate the total quantity of Phloxine B present in both phases for each test (A, B) (show your calculations) Compare the above with the amount of Phloxine B originally introduced in each separatory funnel (A, B) The partition of this substance depends on the pH. Comment on how you can determine whether there is ionized and non-ionized form in your two test conditions (two tests A and B) Later in CAL: You will see the group overall mean logD values and you will compare your individual logD values to this.

Test report and Lab Questions Prepare a lab report including the following information: (i) Name of the substance, and relevant chemical properties. (ii) Temperature of the determination. (iii) Concentration of stock solution and volume (amount) added to separatory funnel. (iv) Data on the analytical procedure used in determining concentrations. Use a table to summarize your results (sample table provided): 6

(v) The measured concentrations in both phases for each determination. (vi) The volume of each phase employed in each test funnel, and the total calculated amount of test substance present in each phase after equilibration. (vii) The calculated values of the logD for each test. Answer the following questions: 1. Based on the results of your analysis, is the substance you tested water-soluble or lipid soluble? Explain your answer. 2. Does this substance have the potential to bioaccumulate? Explain your answer. 3. Based on this experience, how long would it take to test 1,000 substances for this one property? Table Results summary Concentration (units) A o w B o w Amount (units) Total amount (units) D logD

Other References: 1. Levitan, H., Food, drug, and cosmetic dyes: biological effects related to lipid solubility. Proc Natl Acad Sci U S A, 1977. 74(7): p. 2914-8.