TEST METHOD Effective: 15 July 2010 Supersedes: DOWM 100931-ME91A DOWM 100931-E10B Ethoxyl Substitution in ETHOCEL TM Ethylcellulose Polymers 1. Scope This method is applicable to the determination of ethoxyl substitution in ETHOCEL TM ethylcellulose polymers over a concentration range of 40-55%, w/w (Note 15.1). This method describes the determination of the ethoxyl content of ethylcellulose according to the USP 32 NF 27 monograph Ethylcellulose, section Assay. 2. Principle When ethylcellulose polymer is reacted with hydriodic acid, one mole of ethyl iodide is liberated for each mole of ethoxyl substituted on the cellulose chain. The resulting ethyl iodide is extracted with o- xylene and analyzed by gas chromatography using flame ionization detection. Quantitation is made by peak area measurements using internal standard calculations and a chromatography data system. 3. Safety 3.1 Each analyst must be acquainted with the potential hazards of the equipment, reagents, products, solvents and procedures before beginning laboratory work. SOURCES OF INFORMATION INCLUDE: OPERATION MANUALS, MATERIAL SAFETY DATA SHEETS, LITERATURE AND OTHER RELATED DATA. Safety information should be requested from the supplier. Disposal of waste materials, reagents, reactants and solvents must be in compliance with applicable governmental requirements. 3.2 Appropriate safety precautions must be taken during handling of hydriodic acid. Refer to the current MSDS for the most up-to-date safety guidelines (Section 3.1). 3.3 Personal protective equipment to be worn during the procedure includes chemical goggles and acid-resistant gloves. If the gloves become contaminated, dispose of them immediately. All work with hydriodic acid must be done in a fume hood and all samples should be stored in the hood. 3.4 During the reaction, the glass vials are under pressure at high temperature. Exercise caution in handling the vials and conduct the reaction and agitation behind a safety shield and in a fume hood. TM Trademark of The Dow Chemical Company ("Dow") or an affiliated company of Dow Page 1 of 11 Copyright 2010 The Dow Chemical Company, All rights reserved
15 July 2010 Page 2 of 11 DOWM 100931-E10B 4. Interferences 4.1 Any ether, ester, or alcohol will react under the conditions of the Zeisel derivatization to form the corresponding iodide. Depending on the component, there is a chance that it could co-elute with ethyl iodide and influence the final result. These types of materials are not known to be present in ETHOCEL ethylcellulose polymers at other than trace levels. 4.2 If results are suspect based on the analytical history of the product, the data should be confirmed using an alternate method. 5. Apparatus (Note 15.2) 5.1 Gas chromatograph: Agilent model 6890, equipped with a flame ionization detector and a packed column injection port, available from Agilent Technologies, Inc., 5301Stevens Creek Blvd, Santa Clara, CA 95051, or equivalent. 5.2 Gas chromatographic column: preconditioned 1/8-inch (3.2-mm) OD, 2-mm ID 5.0-m stainless steel column packed with 3% G2 on 150-µm to 180-µm mesh support S1A, available from Ohio Valley Specialty Company, 115 Industry Road, Marietta, Ohio 45750. Note: If the column is not preconditioned, it is suggested that preconditioning be done whenever a new column is installed (Appendix I). 5.3 Chromatography data system: Agilent EZChrom, available from Agilent Technologies, Inc., or equivalent. 5.4 Autosampler: Agilent model 7683B, available from Agilent Technologies, Inc., or equivalent. 5.5 Autosampler syringe: 10-µL, 23-gauge, for use with the Agilent model 7683B autosampler, available from Agilent Technologies, Inc., or equivalent. 5.6 Syringes: 100-µL, Hamilton No. 710N, gas-tight syringes, available from Fisher Scientific, 2000 Park Lane Drive, Pittsburgh, PA 15275, or equivalent. 5.7 Syringes: 3-mL disposable syringes, B-D order number 309582, available from Fisher Scientific, 2000 Park Lane Drive, Pittsburgh, PA 15275, or equivalent. 5.8 Heating block: with a tight fitting lid to contain heat in the unit, with wells measuring 24- mm diameter 45-mm to hold serum vials, capable of maintaining a temperature of 125 C, fabricated in-house, The Dow Chemical Company, Midland, MI 48674 (Note 15.3). Note: Reacti-Therm I module, part number 18800, with Reacti-Block Q, part number 18814, containing eight 26-mm diameter 46-mm sample wells, available from Fisher Scientific is a commercially available equivalent heating block. The Reacti-Block uses magnetic stir bars so that the reciprocal shaker (Section 5.12) is unnecessary. 5.9 Serum vials (reaction vials): 10-mL, 23-mm o.d. 46-mm high with a 20-mm o.d. and 13- mm i.d. opening head space vials, Catalog number 03-340-75B, available from Fisher Scientific, or equivalent (Notes 15.4 and 15.5).
15 July 2010 Page 3 of 11 DOWM 100931-E10B 5.10 Septa: for serum vials, 20-mm polytetrafluoroethylene (PTFE)-faced gray butyl rubber, Wheaton part number 224168, available from Fisher Scientific, or equivalent. 5.11 Aluminum crimp-seals: for serum vials, 20-mm, available from Fisher Scientific, or equivalent. 5.12 Crimping tool: for 20-mm crimp-seals, available from Fisher Scientific, or equivalent. 5.13 Reciprocal shaker: capable of at least 100 oscillations per minute, Eberbach model 6000, available from VWR International, 1310 Goshen Parkway, West Chester, PA 19380, or equivalent. 5.14 Electronic balance: capable of weighing to 0.0001 g, Mettler model AE163, available from Mettler-Toledo Inc., 1900 Polaris Parkway, Columbus, OH 43240, or equivalent. 5.15 Electronic pipetter: Eppendorf, capable of dispensing 2 ml, available from Fisher Scientific, or equivalent. 5.16 Pipette tips: for use with Eppendorf pipetter (Section 5.14), 5000-µL, part number 22 49 208-0, available from Fisher Scientific, or equivalent. 5.17 GC vial inserts: catalog number 03-375-3B, available from Fisher Scientific, or equivalent. 5.18 GC vials: 1.8-mL size with crimp or snap caps, available from Fisher Scientific, or equivalent. 5.19 Volumetric flask: 250-mL, available from Fisher Scientific, or equivalent. 5.20 Bottle: 250-mL, glass bottle with PTFE-lined cap, available from Fisher Scientific, or equivalent. 5.21 Oxygen removal system: suggested for use with the helium carrier gas supply, high capacity gas purifier, available from Supelco, 3050 Spruce St, St Louise, MO 63103, or equivalent. 6. Reagents 6.1 o-xylene: reagent grade, available from Fisher Scientific, or equivalent. 6.2 Toluene: 99+% purity, ACS grade, available from Sigma-Aldrich Corporation, 3050 Spruce Street, St. Louis, MO 63103, or equivalent. 6.3 Iodoethane (ethyl iodide): 99% purity, available from Sigma-Aldrich Corporation, or equivalent. Note: The purity of this standard should be provided by the supplier for use in Section 11. 6.4 Adipic acid: certified grade with a melting point of 151-153 C, available from Fisher Scientific, or equivalent. 6.5 Hydriodic acid: 57% purity, specific gravity 1.69 to 1.70, boiling point 127 C, stabilized with hypophosphorous acid, available from Sigma-Aldrich Corporation, or equivalent (Note 15.6).
15 July 2010 Page 4 of 11 DOWM 100931-E10B 6.6 Reference material (optional): a batch of ETHOCEL ethylcellulose polymer, segregated and retained, available from The Dow Chemical Company, Customer Service Center, 2040 Building, W.H. Dow Center, Midland, MI 48674. 7. Reagent Solution 7.1 Internal standard solution Note: The shelf life of this solution is up to 30 days from the date of preparation if the solution is sealed effectively to prevent evaporation. Evaporation can cause the concentration of toluene to decrease, leading to false high values for substitution. It is also recommended that no more than 80% of the total solution be used unless it is used within one week from the date of preparation. 7.1.1 Into a 250-mL volumetric flask, pipette 3.00 ml toluene (Section 6.2). 7.1.2 Dilute the contents of the flask to volume with o-xylene (Section 6.1). Cap the flask and mix well. 7.1.3 Transfer to a glass bottle and cap. This solution may be stored in a refrigerator for up to one month. 8. Analysis Conditions Note: The parameters summarized below were used in the validation of the method. Pressures, flow rates, and integrator parameters will depend on each chromatographic system and may differ from those stated below. Instrument: Agilent model 6890 GC/FID, EZChrom CDS, model 7683B autosampler Column: 2-mm 5.0-m stainless steel column packed with 3% G2 on 150-µm to 180-µm mesh support S1A, pre-conditioned Detector: Flame Ionization Detector Temperatures: Injector: 200 C Detector: 200 C Oven: 80 C (7 min) - 30 C/min - 200 C (2 min hold) (Note 15.7) FID Flows: H 2 : 40 ml/min Air: 200 ml/min Injection Volume: 1-µL, automated Inlet mode: Splitless Autosampler parameters: # of Sample washes: 5 # of Pumps: 5 Viscosity: 0 # of Solvent A washes: 5 (o-xylene) # of Solvent B washes: 5 (acetone) Approximate retention times Ethyl iodide 2.5 minutes
15 July 2010 Page 5 of 11 DOWM 100931-E10B Toluene (IS) o-xylene (solvent) 3.5 minutes 6.8 minutes A representative sample chromatogram is illustrated in Figure 1. 9. Calibration 9.1 Condition the GC column prior to calibration as follows: 9.1.1 Conduct a blank analysis (with no injection) to take the column through the heating steps. If the baseline appears to be stable, continue on to Section 9.2.2. 9.1.2 Conduct a primer analysis of a previously or newly prepared sample. Discard the data from this analysis it is for column conditioning purposes only. 9.2 Calibration Standard (Note 15.8) 9.2.1 Tare a serum vial on the balance and add 100 to 110 mg of adipic acid (Section 6.4). Using an Eppendorf pipetter, pipette 4 ml of internal standard solution (Section 7.1) and 4 ml of hydriodic acid (Section 6.5) into the serum vial. Place a 20-mm septum on the vial and crimp securely. 9.2.2 Tare the vial and using a 100-µL syringe, add 50 µl of ethyl iodide (Section 6.3) through the septum. Weigh the vial and record the amount of ethyl iodide added to the nearest 0.0001 g. Mix the contents by shaking briefly. 9.2.3 Using a disposable syringe, pierce the septum and remove approximately 1 ml of the top layer. 9.2.4 Place GC vial inserts into each of two GC vials and fill each approximately 80% full with the aliquot from Section 9.2.3. Immediately cap the vials. 9.3 Analyze the calibration standards (Section 9.2) using the conditions summarized in Section 8. 10. Procedure (Note 15.9) 10.1 Prepare the sample solution as follows: 10.1.1 Determine the loss on drying of the sample according to DOWM 100810 (Reference 16.1). 10.1.2 Weigh (and record to the nearest 0.0001 g), 50 mg ± 5 mg of the sample into a serum vial. Tare the vial. 10.1.3 Add 60 ± 5 mg adipic acid and record the weight to the nearest 0.001 g (Note 15.10). 10.1.4 Using an electronic pipetter, add 2 ml of the internal standard solution (Section 7.1) to the vial in Section 10.1.3.
15 July 2010 Page 6 of 11 DOWM 100931-E10B 10.1.5 In a fume hood, using an electronic pipetter, add 2 ml of hydriodic acid into the vial in Section 10.1.4. Use a crimping tool to crimp seal the vial with the 20-mm septum and an aluminum seal. 10.1.6 Tare the balance, weigh the vial and record the weight to the nearest 0.0001 g. 10.1.7 Mount the heating block (Section 5.7) on the reciprocal shaker (Section 5.12) and place in a fume hood. Equilibrate the heating block at 125 C. 10.1.8 Place the vial (Section 10.1.6) in the heating block and heat at 125 C for 60 minutes, shaking continuously at a rate of at least 100 oscillations per minute. 10.1.9 After the 60 minute heating and shaking time, remove the vial from the heating block and cool to room temperature for approximately 45 minutes. 10.1.10 Weigh the vial and compare the weight to the original weight obtained in Section 10.1.6. If the loss is greater than 10 mg, discard the mixture and prepare another vial repeating Sections 10.1.2 to 10.1.10. 10.2 The contents of the vial will separate into two layers. With a disposable syringe, pierce the septum and remove approximately 0.5 ml of the top layer. 10.3 Place a GC vial insert in a GC vial and fill approximately 80% full with the aliquot from Section 10.2. Cap the vial. 10.4 Analyze the sample solution (Section 10.3) using the conditions outlined in Section 8. 10.5 Prior to reporting results from a run, it is recommended that the reference material assay results (Section 15.9) be checked and shown to be within stated tolerance. 11. Calculation Calculate the percentage of ethoxyl substitution in the original sample as follows: VIS,SS 45.1 Area EI,SS Area IS,CS m EI,CS PEI % ethoxyl = 100% V IS,CS 156 Area IS,SS Area EI,CS msample (100% d)/100% Where: % ethoxyl = concentration (%, w/w) of ethoxyl substitution in the ETHOCEL ethylcellulose polymer sample V IS,SS = volume (ml) of internal standard solution added to the sample solution (Section 10.1.4) V IS,CS = volume (ml) of internal standard solution added to the calibration standard (Section 9.2.1) 45.1 = formula weight (g/mol) of ethoxyl (-OC 2 H 5 ) 156 = formula weight (g/mol) of ethyl iodide (-IC 2 H 5 ) Area EI,SS = ethyl iodide peak area in the chromatogram obtained from the analysis of the sample solution (Section 10.4)
15 July 2010 Page 7 of 11 DOWM 100931-E10B Area IS,CS = average internal standard peak area in the chromatogram obtained from the analysis of the calibration standard (Section 9.3) m EI,CS = mass of ethyl iodide (mg) used in the calibration standard (Section 9.2.2) P EI = purity of the ethyl iodide used in the preparation of the calibration standard in decimal form (Section 6.3) Area IS,SS = internal standard peak area in the chromatogram obtained from the analysis of the sample solution (Section 10.4) Area EI,CS = average ethyl iodide peak area in the chromatogram obtained from the analysis of the calibration standard (Section 9.3) m sample = mass (mg) of sample used to prepare the sample solution (Section 10.1.2) d = loss on drying as a percentage (Section 10.1.1). 12. Precision Precision data determined from multiple analyses [n] of samples from four product families of ETHOCEL ethylcellulose polymer determined by this procedure are given below. The analyses were performed over a two-day period. The stated prediction intervals at the 95% confidence level [ ± t (n 1) s ; where t (n-1) = t-value at n-1 degrees of freedom, and s = standard deviation of the validation data] relate to future final results determined on similar samples. This assumes a normal distribution of results and equal variability between locations. The distributions of the results are assumed to be normal. The validity of this assumption has been verified using the Shapiro-Wilk test for normality. The test confirmed that the results could originate from normal distributions. Sample n t (n-1) Average x Standard Deviation s Estimated Prediction Interval at the 95% Confidence Level for a future result (%, w/w) (%, w/w) (± %, w/w) S10 12 2.201 48.33 0.14 0.32 M70LDIN 12 2.201 45.51 0.21 0.46 S10FPPR 12 2.201 48.37 0.26 0.56 S100PR 12 2.201 48.49 0.10 0.23 13. Accuracy The accuracy was not determined due to the unavailability of a well-characterized standard of ETHOCEL ethylcellulose polymer. 14. Linearity The response of the system for ethyl iodide was found to be linear over a concentration range equivalent to 6 to 63 %, w/w ethoxyl substitution in ETHOCEL ethylcellulose polymer samples.
15 July 2010 Page 8 of 11 DOWM 100931-E10B 15. Notes 15.1 The applicable range is based on the current range of ethoxyl substitution in ETHOCEL ethylcellulose polymer products. The true applicable range may be larger based on the data determined in the linearity study (Section 14). 15.2 Analytical method performance can be affected by minor differences in instrumentation, reagents, and laboratory technique. Consequently, the method should be qualified in the performing laboratory to confirm its performance and suitability. In addition, analytical instruments should be calibrated at appropriate frequencies. 15.3 When setting the temperature of the heating block, the temperature inside a serum vial must be checked to verify the correct reaction temperature. Use a 5-mL syringe to fill a serum vial with 4 ml of silicone oil and crimp seal the vial. Use a syringe needle (18 gauge or lower) to puncture the septum and insert a thermocouple into the vial until it is just below the surface of the silicone oil. Place the vial in the heating block and wait 30 minutes for the temperature to equilibrate. Make adjustments to the heating module to bring the temperature inside the vial to 125 ± 5 C. 15.4 This method uses 10-mL serum vials in place of the 5-mL serum vials specified by USP (Reference 16.2). 15.5 For serum vials taller than 46-mm, the vial headspace will extend above the top of the heater block, potentially causing the temperature inside the vial to be below the temperature of the heating block. To ensure that the temperature of the vial contents and the heating block temperature are the same, the vial headspace must not extend above the heating block. 15.6 The hydriodic acid should be slightly yellow in color with no precipitate present. Bottles of hydriodic acid should be discarded 3 months after opening. 15.7 A temperature program including a ramp to 200 C is used in place of the isothermal program specified by USP (Reference 16.2) to ensure the elution of heavier components from the column prior to subsequent analyses. 15.8 A new calibration standard should be prepared daily and also whenever a new internal standard solution is prepared. 15.9 Due to the large number of procedural steps involved with this method, it is recommended that statistical quality control (SQC) charting of a reference material of ETHOCEL ethylcellulose polymer (Section 6.6) be used. It is also recommended that this reference material be run with every set of samples. The SQC chart that illustrates these data should be routinely updated. In addition, to verify proper performance of the column, reference materials should not always be run at the beginning of a set of samples. Instead, it is suggested that they be distributed randomly throughout the analysis set to ensure the best accuracy. 15.10 To ensure there is an excess of adipic acid relative to the ETHOCEL ethylcellulose polymer, 60 mg is the target weight of adipic acid instead of 50 mg as specified by USP (Reference 16.2).
15 July 2010 Page 9 of 11 DOWM 100931-E10B 16. References 16.1 DOWM 100810 (latest version), "Loss on Drying of ETHOCEL Ethylcellulose Ethers", available from The Dow Chemical Company, QAIS, 1897 Building, Midland, MI, 48667. 16.2 USP 32 NF 27 monograph Ethylcellulose, available from United States Pharmacopeial Convention, Inc., 12601 Twinbrook Parkway, Rockville, MD 20852. 16.3 Food Chemicals Codex 6, monograph Ethylcellulose, available from United States Pharmacopeial Convention, Inc., 12601 Twinbrook Parkway, Rockville, MD 20852. *************************************************************************************** THE INFORMATION HEREIN IS PRESENTED IN GOOD FAITH, BUT NO WARRANTY, EXPRESS OR IMPLIED, IS GIVEN NOR IS FREEDOM FROM ANY PATENT OWNED BY THE DOW CHEMICAL COMPANY OR BY OTHERS TO BE INFERRED. IN THE HANDS OF QUALIFIED PERSONNEL, THE PROCEDURES ARE EXPECTED TO YIELD RESULTS OF SUFFICIENT ACCURACY FOR THEIR INTENDED PURPOSE; BUT RECIPIENTS ARE CAUTIONED TO CONFIRM THE RELIABILITY OF THEIR TECHNIQUES, EQUIPMENT, AND STANDARDS BY APPROPRIATE TESTS. ANYONE WISHING TO REPRODUCE OR PUBLISH THIS MATERIAL IN WHOLE OR IN PART SHOULD REQUEST WRITTEN PERMISSION FROM THE DOW CHEMICAL COMPANY.
15 July 2010 Page 10 of 11 DOWM 100931-E10B Figure 1. A representative ETHOCEL ethylcellulose polymer sample chromatogram obtained using the analysis conditions listed in Section 7. Volt 30 25 20 15 10 5 Front FID S 962-2 Name Retention Time Area Ethyl Iodide 2.507 102716040 Toluene 3.485 148119415 6.800 15607148087 10.741 10445780 30 25 20 15 10 5 Volt 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Minutes Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow
15 July 2010 Page 11 of 11 DOWM 100931-E10B Appendix I Column Preconditioning 1. Once the column is installed, heat at 200 C for at least 15 minutes to thermally condition and remove any contaminants. 2. Conduct a blank analysis (with no injection) using the analysis conditions outlined in Section 8. A flat baseline should result. 3. Chemically condition the column by injecting 5 µl of a 50:50 (v/v) ethyl iodide:o-xylene solution using the analysis conditions outlined in Section 8. Repeat this conditioning 3 to 5 times. 4. Analyze a previously prepared sample of known composition. Adjust the retention times in the existing calibration table and evaluate the results to determine if further conditioning is required recognizing that the new column has not been calibrated. For example, if a reference sample is used, the results should be within the lab established control limit. 5. Proceed to Section 9 to calibrate the column.