TEST METHOD. Methoxyl and Hydroxypropoxyl Substitution in Cellulose Ethers by Zeisel Gas Chromatography

Transcription

1 TEST METHOD Effective: 10 February 2015 DOWM E15E Supersedes: DOWM E12D Methoxyl and Hydroxypropoxyl Substitution in Cellulose Ethers by Zeisel Gas Chromatography 1. Scope 1.1 This method is applicable to the determination of the following substitutions in cellulose ether products over the listed weight percent ranges: Component Applicable Range (Note 17.1) (% w/w) Methoxyl substitution Hydroxypropoxyl substitution This method describes the determination of the methoxyl content of methylcellulose and the methoxyl and hydroxypropoxyl content of hydroxypropyl methylcellulose according to the current USP, PhEur and JP Methylcellulose and Hypromellose monographs Assay sections (References 18.1 to 18.3). The methoxyl and hydroxypropoxyl substitutions determined by this method are equivalent to those determined according to the current Food Chemicals Codex, Methylcellulose and Hydroxypropyl methylcellulose monograph (Reference 18.4). 2. Principle 3. Safety 2.1 When methylcellulose or hydroxypropyl methylcellulose are reacted with hydriodic acid in the presence of adipic acid, one mole of methyl iodide is liberated for each mole of methoxyl substituent on the cellulose chain and one mole of isopropyl iodide is liberated for each mole of hydroxypropyl substituent on the cellulose chain. The methyl iodide and isopropyl iodide are extracted in-situ with o-xylene and analyzed by gas chromatography using thermal conductivity or flame ionization detection. Quantitation is made using internal standardization. 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 Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow Page 1 of 18 Copyright 2015 The Dow Chemical Company. All rights reserved. THE INFORMATION HEREIN IS PRESENTED IN GOOD FAITH, BUT NO WARRANTY, EXPRESS OR IMPLIED, IS GIVEN NOR IS FREEDOM FROM INFRINGEMENT OF 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.

2 requested from the supplier. Disposal of waste materials, reagents, reactants and solvents must be in compliance with applicable governmental and company requirements. 3.2 Appropriate safety precautions must be taken during handling of hydriodic acid. Review the Material Safety Data Sheet (MSDS) for this material prior to working with this method (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. 4. Interferences 4.1 This method is not specific for cellulose ethers because any alkyl ether will react under these conditions to form the respective alkyl iodide. The only compounds which will directly interfere with the analysis are those which produce methyl or isopropyl iodide, because other iodides will elute at different times during the gas chromatographic analysis. Examples of materials which will interfere are hydroxypropyl cellulose, polypropylene glycol and methyl ethers of propylene glycol. These types of compounds are not known to be present in typical methylcellulose or hydroxypropyl methylcellulose samples except in trace quantities. The exception to this is for surface-treated hydroxypropyl methylcellulose products, which must be analyzed before surface-treating to obtain accurate Zeisel substitution test results. 4.2 If results are suspect based on the analytical history of a product, the results should be confirmed using an alternate method. 5. Apparatus (Note 17.2) 5.1 Gas chromatograph: Agilent model 6890, equipped with a thermal conductivity or flame ionization detector, with EZChrom software, available from Agilent Technologies, Inc., 5301 Stevens Creek Blvd, Santa Clara, CA 95051, or equivalent. 5.2 Autosampler: Agilent 7683, available from Agilent Technologies, Inc., or equivalent. 5.3 Gas chromatographic column (Note 17.3): Packed column: 3- to 4-mm 1.8- to 3-m glass column packed with 20% liquid phase G1 on mesh support S1D that is not silanized, pre-conditioned, available from Grace Davison Discovery Sciences, 2051 Waukegan Road, Deerfield, IL , or equivalent. Or Note: If the column is not pre-conditioned, it is suggested that pre-conditioning be done whenever a new column is installed (Appendix I) Capillary column: ZB-5, 30-m 0.53-mm 5.0-µm available from Phenomenex, or equivalent. Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow Page 2 of 18

3 5.4 GC autosampler vials: with 400-µL glass inserts, available from Agilent Technologies, Inc., or equivalent. 5.5 Reaction vials (Note 17.4): mL, 23-mm o.d. 46-mm high with a 20-mm o.d. and 13-mm i.d. opening headspace vials, catalog number B, available from Fisher Scientific, 2000 Park Lane Drive, Pittsburgh, PA or equivalent. Or mL, 20 mm o.d. 38-mm high with a 20-mm o.d. and 13-mm i.d. opening headspace vials, catalog number , available from Macherey & Nagel, or equivalent. 5.6 Septa and crimp seal rings for reaction vials: For 10-mL vials: 20-mm gold top crimp seals with polytetrafluoroethylene (PTFE)-faced butyl rubber septa, available from Alltech Associates, or equivalent. Or For 5-mL vials: 20-mm aluminum crimp seals with polytetrafluoroethylene (PTFE)-faced silicone septa, catalog number , available from Agilent Technologies, Inc., or equivalent. 5.7 Crimping tool: for 20-mm seal rings, available from Fisher Scientific, or equivalent. 5.8 Reciprocal shaker: capable of 100 oscillations per minute, Eberbach model 6000, available from VWR International, 1310 Goshen Parkway, West Chester, PA 19380, or equivalent. 5.9 Analytical balance: capable of weighing to g, Mettler model AE163, available from Mettler-Toledo Inc., 1900 Polaris Parkway, Columbus, OH 43240, or equivalent Syringes: 25-L, Hamilton No. 702N, available from Fisher Scientific, or equivalent Syringes: 50-µL, Hamilton No. 705N, gas-tight, available from Fisher Scientific, or equivalent Heating block (Note 17.5): with a secure lid to contain heat in the unit, capable of maintaining a temperature of 130 C, and mounted on a reciprocal shaker (Section 5.8). The diameter of the wells in the heating block should be sufficient to accept the reaction vials while minimizing space between the walls of the vial and the well. The depth of the wells in the heating block should contain most of the height of the reaction vials to ensure the entire vial maintains a temperature of 130 C during the reaction For 10-mL reaction vials: Heating block with wells measuring 24-mm diameter 45-mm depth, fabricated in-house, The Dow Chemical Company, Midland, MI 48674, or equivalent For 5-mL reaction vials: Heating block with wells measuring 21-mm diameter 31-mm to hold reaction vials, available from Liebisch, catalog number , or equivalent. Note: A Thermo Scientific Reacti-Therm unit with magnetic stir bars may be used instead of a heating block mounted on a reciprocal shaker. Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow Page 3 of 18

4 For 10-mL reaction vials: Thermo Scientific Reacti-Therm I module, part number with Reacti-Block Q, part number 18814, containing eight 26-mm diameter 46-mm sample wells, available from VWR International. For 5-mL reaction vials: Thermo Scientific Reacti-Therm I module, part number with Reacti-Block B, part number 18802, containing nine 21-mm diameter 32-mm sample wells, available from VWR.International Oxygen removal system: suggested for use with the helium carrier gas supply, high capacity gas purifier, available from Supelco, 3050 Spruce St., St. Louis, MO 63103, or equivalent Electronic pipette: 0.1- to 50-mL, Eppendorf Multipette stream, available from Fisher Scientific, or equivalent Drying tin: metal tin, available from Fisher Scientific, or equivalent Syringes: 3-mL disposable syringes, B-D order number , available from Fisher Scientific, or equivalent Volumetric flasks: 1000-mL, available from Fisher Scientific, or equivalent Oven: capable of maintaining a temperature of C, available from Fisher Scientific, or equivalent Desiccator: with indicating desiccant, available from Fisher Scientific, or equivalent Autosampler syringe: 10-µL, PTFE-tipped, part number NS604403, available from Fisher Scientific, or equivalent. 6. Reagents Note: The highest purity reagents available should be purchased for use with this method. Certificates of analysis including sample purity are available from the manufacturer. 6.1 Adipic acid: certified grade with a melting point of C, available from Fisher Scientific, or equivalent. 6.2 o-xylene: reagent grade, available from Fisher Scientific, or equivalent. 6.3 n-octane: 99+% purity, Gold Label grade, available from Sigma-Aldrich Corporation, 3050 Spruce Street, St. Louis, MO 63103, or equivalent. 6.4 Iodomethane (methyl iodide): 99+% purity, available from Sigma-Aldrich Corporation, or equivalent Iodopropane (isopropyl iodide): 99+% purity, available from Sigma-Aldrich Corporation, or equivalent. 6.6 Hydriodic acid: 57% purity, specific gravity of 1.69 to 1.70, boiling point 127 C, stabilized with hypophosphorous acid, available from Aldrich Chemical Company, Inc., or equivalent (Note 17.6). 6.7 Reference material (optional): a batch of METHOCEL cellulose ether, segregated and retained, available from The Dow Chemical Company, Customer Service Center, 2040 Building, W.H. Dow Center, Midland, MI Acetone: reagent grade, available from Fisher Scientific, or equivalent. Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow Page 4 of 18

5 7. Reagent Solutions 7.1 Internal standard solution (containing approximately 30 mg/ml n-octane) (Note 17.7) 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 n-octane 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 Tare a 1000-mL volumetric flask on the analytical balance. Add approximately 350 g of o-xylene to the flask. Tare the flask Add 30 5 g of n-octane to the flask and record weight added to the nearest g. Re-tare the flask Dilute the contents of the flask to the mark with o-xylene. Cap the flask and mix thoroughly Calculate the concentration of n-octane in the internal standard solution as follows: Wn octane Pnoctane 1000 CIS Vtotal where: C IS = concentration (mg/ml) of n-octane in the internal standard solution W n-octane = weight (g) of n-octane added to the internal standard solution (Section 7.1.2) P n-octane = purity of n-octane expressed as a decimal (Section 6.3) 1000 = conversion from g to mg V total = total volume (ml) of the internal standard solution (Section 7.1.3) 8. Analysis Conditions Notes: 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. The parameters listed below are specific for the use of a packed column. For use of the capillary column choose appropriate parameters (Note 17.3). Gas chromatograph: Agilent model 6890 with EZChrom software Detector: Thermal Conductivity (TCD) or Flame Ionization (FID) TCD sensitivity: Low Range (TCD): 1 Range (FID): 5 Column: 3- to 4-mm 1.8- to 3-m glass column packed with 20% liquid phase G1 on mesh support S1D that is not silanized, pre-conditioned Autosampler: Agilent Model 7683 Temperatures: Oven: 100 C isothermal for 15 minutes, then ramp to 200 C and hold for 15 minutes to remove any residue from the column Injector: 200 C Detector: 250 C Flows: Carrier gas: 20 ml/minute, helium Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow Page 5 of 18

6 TCD Reference: 60 ml/minute, helium, with a make-up flow of 3 ml/min FID Air: 280 ml/minute FID Hydrogen: 40 ml/minute Injection volume: 1.0 µl, automated Integration parameters: Minimum area: 500 Peak Width: 0.2 Threshold: 250 Calibration: internal standard Reference peaks: methyl iodide, isopropyl iodide, and n-octane Autosampler parameters: # of Sample washes: 5 # of Pumps: 5 Viscosity: 0 # of Solvent A washes: 5 (o-xylene) # of Solvent B washes: 5 (acetone) 8.1 Representative calibration standard and sample chromatograms are illustrated in Figures 1 through Calibration (Note 17.7) 9.1 Calibration standard preparation: Weigh 85 5 mg of adipic acid into a reaction vial (Note 17.9) Using an electronic pipette, add 2.0 ml of 57% hydriodic acid into the vial. Tare the vial on the analytical balance Using an electronic pipette, add 2.0 ml of internal standard solution into the tared vial (Note 17.7). Crimp seal the vial securely and check the seal by hand twisting the cap to make sure it does not move and is thus sealed securely Re-tare the vial. Using a 25-L syringe, add 15 L of isopropyl iodide through the septum of the vial. Record the weight (m PI,CS ) of isopropyl iodide in mg to the nearest 0.1 mg Re-tare the vial. Using a 50-L gas-tight syringe, add 45 L of methyl iodide through the septum of the vial. Record the weight (m MeI,CS ) of methyl iodide in mg to the nearest 0.1 mg. Mix the contents by shaking briefly Repeat Section to to prepare a second calibration standard Determine the weight (g) of internal standard in the calibration standard solution as follows: CIS VISS,CS WIS,CS 1000 where: W IS,CS = weight (g) of internal standard in the calibration standard solution C IS = concentration (mg/ml) of n-octane in the internal standard solution (Section 7.1.4) V ISS,CS = volume (ml) of internal standard solution added to the calibration standard solution (Section 9.1.3) 1000 = conversion from mg to g Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow Page 6 of 18

7 9.2 Condition the GC column prior to calibration as follows: 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 Conduct a primer analysis of a previously or newly prepared sample. Discard the data from this analysis it is for column conditioning purposes only. 9.3 For each calibration standard solution, transfer the top layer using a disposable syringe into a GC autosampler vial with a 400-µL insert. Crimp seal the GC vial. 9.4 At the beginning of each sample analysis sequence analyze one of the prepared calibration standard solutions by injecting duplicate 1-L aliquots and analyzing according to conditions outlined in Section 8. At the end of the sample analysis sequence analyze the second of the prepared calibration standard solutions by injecting duplicate 1-L aliquots and analyzing according to conditions outlined in Section Calculate the relative response factor for each injection of the two calibration standard solutions as follows: Area RRFi Area IS,j i,j Wi,j Pi V IS,j where: RRF i = relative response factor (mg/ml) for the component of interest i = component of interest (methyl iodide or isopropyl iodide) Area IS,j = area counts for the internal standard peak obtained from the analysis of calibration standard solution j (Section 9.4) j = calibration standard solution of interest Area i,j = area counts for the component of interest from the analysis of calibration standard solution j (Section 9.4) W i,j = weight (mg) of the component of interest added to calibration standard solution j (Section and 9.1.5) P i = purity of the component of interest expressed as a decimal (Section 6.4 or 6.5) V IS,j = volume (ml) of the internal standard added to calibration standard solution j 9.6 Calculate the average relative response factors obtained from the four injections of the calibration standard solutions. 10. Procedure 10.1 Sample and reference material preparation: Transfer approximately 2 g of sample into a drying tin. Place the tin in an oven equilibrated at C for at least 60 minutes prior to its use. Remove the tin from the oven, immediately cap with the tight-fitting lid, and place in a desiccator to cool to room temperature before proceeding to Section Tare a reaction vial on an analytical balance. Transfer 65 5 mg of dried sample into the tared vial. Record the weight to the nearest 0.1 mg. Re-tare the vial Add 85 5 mg of adipic acid to the reaction vial (Note 17.9). Record the weight to the nearest 0.1 mg Pipet 2.0 ml of internal standard solution into the vial using an electronic pipette (Note 17.7). Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow Page 7 of 18

8 In a fume hood, carefully pipet 2.0 ml of 57% hydriodic acid into the vial. Use a crimping tool to crimp seal the vial securely, double check by hand twisting the cap to make sure it does not move and is thus sealed securely Re-tare the analytical balance. Place the filled and sealed vial on the balance and record the total weight to the nearest 0.1 mg Mount the heating block on a reciprocal shaker (located behind a safety shield in a fume hood) and preheat the heating block to C (Notes 17.5 and 17.8) Place the reaction vial containing the sample solution in the preheated heating block and shake continuously at approximately 100 oscillations per minute for 60 minutes At the completion of the 60-minute heating step, remove the sample and cool to room temperature. The sample solution will separate into two distinct layers Re-weigh the sample vial and record the total weight to the nearest 0.1 mg. If there is any evidence of a leak or if there is a weight loss of greater than 10 mg, the vial must be discarded and a new sample solution prepared Determine the weight (g) of internal standard in the sample solution as follows: C IS VISS,sample WIS,sample 1000 where: W IS,sample = weight (g) of internal standard in the sample solution C IS = concentration (mg/ml) of internal standard in the internal standard solution (Section 7.1.4) V ISS,sample = volume (ml) of internal standard solution added to the sample solution (Section ) 1000 = conversion from mg to g 10.2 Using a disposable syringe, transfer an aliquot of the top layer of the sample solution (Section 10.1) into the 400-µL insert in a GC autosampler vial. Crimp seal the GC vial Inject a 1-µL aliquot from the GC vial into the gas chromatograph and analyze according to the chromatographic conditions summarized in Section Prior to reporting results from a run it is recommended that the reference material results (Note 17.10) be checked and shown to be within established control limits. 11. System Suitability Note: It is recommended that system suitability be monitored on a periodic basis using a sample containing both methoxyl and hydroxypropoxyl substitution Prepare the sample solution (or reference material solution) as outlined in Section 10.1 and 10.2 or the calibration standard solution as outlined in Section 9.1 and Inject a 1-µL aliquot of the top layer of the solution in Section 11.1 into the gas chromatograph, and separate according to the chromatographic conditions summarized in Section 8. Repeat the analysis four additional times with the same sample solution (a total of five chromatograms should be obtained) Conduct the calculations outlined below using data from the five chromatograms obtained in Section Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow Page 8 of 18

9 Determine the peak tailing factor for each peak of interest (methyl iodide, isopropyl iodide, and the internal standard, n-octane) as outlined below. Wi;0.05 Ti (2 f) where: T i = peak tailing factor for the peak of interest i = peak of interest (methyl iodide, isopropyl iodide, or the internal standard, n-octane W i; 0.05 = peak width (minutes) at 5% peak height (Section 11.2) f = width (minutes) between the peak maximum and the front edge of the peak at 5% of the peak height (Section 11.2) Determine the average peak tailing factor and the % relative standard deviation (% RSD) for each peak of interest using the equations provided below. The average peak tailing factor is acceptable if it is between 0.9 and 1.5. The RSD is acceptable if is less than or equal to 3.0%. Avgi Ti SD %RSD i i Avgi where: Avg i = average peak tailing factor for the peak of interest i = peak of interest (methyl iodide, isopropyl iodide, or the internal standard, n-octane) T i = peak tailing factor for the peak of interest (Section ) % RSD i = % relative standard deviation of the 5 peak tailing factors obtained for the peak of interest SD i = standard deviation of the 5 peak tailing factors obtained for the peak of interest For each peak of interest calculate the adjusted peak area ratio for each injection. Area Ratioi Area i,sample IS,sample W IS, sample W sample where: Ratio i = adjusted peak area ratio for the peak of interest calculated from the sample Area i,sample = peak area for the peak of interest obtained during analysis of the sample (Section 11.2) i = peak of interest (methyl iodide or isopropyl iodide) W IS,sample = weight (g) of internal standard (n-octane) in the sample solution (Section ) Area IS,sample = peak area for the internal standard (n-octane) obtained during analysis of the sample (Section 11.2) W sample = weight (mg) of sample in the sample solution (Section ) Notes: Use W IS,CS instead of W IS,sample if the calibration standard solution is used for the system suitability test (Section 9.1.7) Use m PI,CS (Section 9.1.4) or m MeI,CS (Section 9.1.5) instead of W sample if the calibration standard solution is used for the system suitability test. Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow Page 9 of 18

10 12. Calculations For each peak of interest calculate the relative standard deviation of the adjusted peak area ratio (Ratio i ) over all injections. If the relative standard deviation does not exceed 2.0%, the chromatographic system is fit for use If one or more of these requirements are not met, complete instrument maintenance or troubleshooting of the instrument and repeat Section If manual calculations are used, determine the concentration (% w/w) of methoxyl substitution in the original sample as follows: 31 AreaMeI,SS averagerrfi VIS,sample %methoxyl 100% 142 AreaIS,SS m sample where: % methoxyl = concentration (% w/w) of methoxyl substitution in the cellulose ether sample 31 = formula weight (g/mol) of methoxyl (-OCH 3 ) 142 = formula weight (g/mol) of methyl iodide (ICH 3 ) Area MeI,SS = methyl iodide peak area in the chromatogram obtained from the analysis of the sample solution (Section 10.3) average RRF i = average relative response factor (mg/ml) for the component of interest (Section 9.6) i = methyl iodide V IS,sample = volume (ml) of internal standard added to the sample solution (Section ) Area IS,SS = internal standard peak area in the chromatogram obtained from the analysis of the sample solution (Section 10.3) m sample = mass (mg) of sample used to prepare the sample solution (Section ) 12.2 If manual calculations are used, determine the concentration (% w/w) of hydroxypropoxyl substitution in the original sample as follows: 75 Area %HP 170 PI,SS averagerrf V Area IS,SS m i sample IS,sample 100% where: % HP = concentration (% w/w) of hydroxypropoxyl substitution in the cellulose ether sample 75 = formula weight (g/mol) of hydroxypropoxyl (-OC 3 H 7 O) 170 = formula weight (g/mol) of isopropyl iodide (IC 3 H 7 ) Area PI,SS = isopropyl iodide peak area in the chromatogram obtained from the analysis of the sample solution (Section 10.3) average RRF i = average relative response factor (mg/ml) for the component of interest (Section 9.6) i = isopropyl iodide V IS,sample = volume (ml) of internal standard added to the sample solution (Section ) Area IS,SS = internal standard peak area in the chromatogram obtained from the analysis of the sample solution (Section 10.3) m sample = mass (mg) of sample used to prepare the sample solution (Section ) Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow Page 10 of 18

11 13. Precision (Notes 17.3, 17.11, 17.12) 13.1 Precision data determined from multiple analyses [n] of five different types of methylcellulose or hydroxypropyl methylcellulose samples are given below. The analyses were performed over an extended period At the 95% confidence level, individual measurements on similar samples may vary from the long-term average by the values shown below [± t (n-1) s; where t (n-1) = t-value at n-1 degrees of freedom, and s = standard deviation of the validation data]. This assumes a normal distribution of results and equal variability between locations The distribution of the results is 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 for methoxyl substitution in the E and F chemistry samples and the results for hydroxypropoxyl substitution in the E, F, and K chemistry samples could originate from normal distributions. The test suggested that the results for methoxyl substitution in the A, J, and K chemistry samples and the results for hydroxypropoxyl substitution in the J chemistry sample did not originate from a normal distribution. The exact nature of the distribution is unknown. Based on Chebychev s Inequality Theorem, 4.47 was used as an estimate for the t-value at the 95% confidence level for the results that do not originate from a normal distribution (Reference 18.5) Methoxyl Substitution Sample n t (n-1) Average Concentration Standard deviation %RSD At the 95% confidence level, individual measurements may vary from the long-term average by: (% w/w) (% w/w) (%) ( % w/w) A, E, F, K, J, Hydroxypropoxyl Substitution 14. Accuracy Sample n t (n-1) Average Concentration Standard deviation %RSD At the 95% confidence level, individual measurements may vary from the long-term average by: (% w/w) (% w/w) (%) ( % w/w) E, F, K, J, The accuracy was not determined due to the unavailability of a well-characterized standard of methylcellulose or hydroxypropyl methylcellulose. Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow Page 11 of 18

12 15. Linearity 15.1 Detector response (using both the thermal conductivity and flame ionization detectors) was found to be linear for iodide components over the ranges stated below The theoretical methoxyl and hydroxypropoxyl substitution for a 65 mg sample are calculated from the concentrations of the iodides in the synthetic solutions and shown below. Component Linear Component Linear Range Range (mg/ml) (% w/w) Methyl iodide Methoxyl substitution equivalent Isopropyl iodide Hydroxypropoxyl substitution equivalent Limits of Detection and Quantitation 17. Notes 16.1 For methoxyl substitution the limit of detection (LOD) and limit of quantitation (LOQ) are expected to be less than the lowest concentration of the linear range (3.2%, w/w). The limits of detection and quantitation were not determined for methoxyl substitution because the applicable range of this analysis is well above the assumed LOD/LOQ The limit of detection (LOD), defined as three times the baseline noise, was determined to be 0.25% (w/w), for hydroxypropoxyl substitution. The limit of quantitation (LOQ), defined as three times the LOD, was determined to be 0.75% (w/w), for hydroxypropoxyl substitution The applicable range is based on current product specifications. The method linearity has been validated over the ranges of mg/ml methyl iodide and mg/ml isopropyl iodide which are equivalent to % (w/w) methoxyl substitution and % (w/w) hydroxypropoxyl substitution, respectively 17.2 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 It has been validated that packed columns and capillary columns produce equivalent test results For reaction vials taller than 45-mm or 31-mm depending on the heating block used, the vial headspace will extend above the top of the heating block (Section 5.12), 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 When setting the temperature of the heating block, the temperature inside a reaction vial must be checked to verify the correct reaction temperature. Use a 5-mL syringe to fill a reaction 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 C. Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow Page 12 of 18

13 17.6 The hydriodic acid should be slightly yellow in color with no precipitate present. Bottles of hydriodic acid should be discarded 3 months after opening The same internal standard solution preparation should be used for all samples and calibration standards solutions. Fresh calibration standard solutions should be prepared whenever the analysis is performed and also whenever a new internal standard solution is prepared The FCC monograph (Reference 18.4) calls for a reaction temperature of 150 C and shaking the vials by hand 3 times during the reaction. The USP (Reference 18.1), PhEur (Reference 18.2) and JP (Reference 18.3) monographs use a reaction temperature of 130 C and continuous shaking. Dow uses a reaction temperature of 130 C and continuous shaking USP specifies a range of 60 to 100 mg for adipic acid. However, the quantity of adipic acid impacts test results. Therefore target a weight of 85 5 mg adipic acid 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 METHOCEL cellulose ether 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 evaluation Precision data were collected using an internal standard solution with a concentration of 2.5% (w/w) of n-octane in o-xylene. This is equivalent to approximately 2.2% (w/v). According to the USP, PhEur, and JP the internal standard solution concentration should be 3.0 mg/ml, equivalent to 3.0% (w/v). This difference in concentration of the internal standard solution is not expected to affect the precision of the method Precision data were collected according to USP30 Hypromellose monograph using a 4-mm 3-m glass column with 20% liquid phase G28 on 100- to 120-mesh support S1C that was not silanized. Parameters for the chromatographic system remained unchanged from USP32 through USP36. Equivalency of these precision data to USP36, 1 st supplement (column: 3- to 4-mm 1.8- to 3-m glass; packed with 20% liquid phase G28 on 100- to 120- mesh support S1D that was not silanized) and USP37 (column: 3- to 4-mm 1.8- to 3-m glass; packed with 20% liquid phase G1 on 100- to 120-mesh support S1D that was not silanized) has been demonstrated. 18. References 18.1 USP, United States Pharmacopeia, monographs Hypromellose and Methylcellulose (current version) available from United States Pharmacopeial Convention, Inc., Twinbrook Parkway, Rockville, MD PhEur, European Pharmacopoeia, monographs Hypromellose and Methylcellulose (current version) available from European Directorate for the Quality of Medicines & HealthCare (EDQM), Council of Europe 7 alleé Kastner, CS 30026, F Strasbourg, FRANCE JP, Japanese Pharmacopoeia, monographs Hypromellose and Methylcellulose (current version) available from Japanese Pharmacopoeia, edited by Society of Japanese Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow Page 13 of 18

14 Pharmacopoeia, published by Yakuji Nippo, Ltd., 1, Kanda Izumicho, Chiyoda-ku, Tokyo, Japan FCC, Food Chemicals Codex, monographs Hydroxypropyl Methylcellulose and Methylcellulose (current version) available from United States Pharmacopeial Convention, Inc., Twinbrook Parkway, Rockville, MD L. J. Bain and M. Engelhardt, Introduction to Probability and Mathematical Statistics, Duxbury Press, Boston, 2 nd Edition, 1992, p. 76. Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow Page 14 of 18

15 Figure 1. A representative chromatogram (Response vs. Time (minutes)) of the calibration standard obtained using the conditions summarized in Section 8. Figure 2. A representative chromatogram (Response vs. Time (minutes)) of a hydroxypropyl methylcellulose E chemistry (hypromellose substitution type 2910) sample obtained using the conditions summarized in Section 8. Response (Volts) Response (Volts) Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow Page 15 of 18

16 Figure 3. A representative chromatogram (Response vs. Time (minutes)) of a hydroxypropyl methylcellulose F chemistry (hypromellose substitution type 2906) sample obtained using the conditions summarized in Section Rear TCD F Robot Std006 Name Retention Time Area Response (mv) Volt Methyl Isopropyl Octane Volt Minutes Figure 4. A representative chromatogram (Response vs. Time (minutes)) of a hydroxypropyl methylcellulose J chemistry (hypromellose substitution type 1828) sample obtained using the conditions summarized in Section Rear TCD WB Name Retention Time Area Response (mv) Volt Isopropyl Octane Volt Methyl Minutes Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow Page 16 of 18

17 Figure 5. A representative chromatogram (Response vs. Time (minutes)) of a hydroxypropyl methylcellulose K chemistry (hypromellose substitution type 2208) sample obtained using the conditions summarized in Section Rear TCD K std 4 Name Retention Time Area Response (V) Volt Methyl Isopropyl Octane Volt Minutes Figure 6. A representative chromatogram of a methylcellulose A chemistry (methylcellulose 2900) sample obtained using the conditions summarized in Section Rear TCD WH28012N Name Retention Time Area 0.5 Volt Response (V) (Methyl-) Isopropyl (1-Octane) Volt Minutes Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow Page 17 of 18

18 Column Preconditioning Appendix I 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) methyl iodide:toluene 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 chromatography data system 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 material is used, the results should be within established control limits. 5. Proceed to Section 9 to calibrate the system. Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow Page 18 of 18