Poloxamer	a	b
124	12	20
188	80	27
237	64	37
338	141	44
407	101	56

Phthalic anhydride–pyridine solution— Dissolve 144g of phthalic anhydride in freshly opened or freshly distilled pyridine containing less than 0.1%of water,and dilute with pyridine to 1000mL.Protect from light,and allow to stand overnight.To verify that thePhthalic anhydride–pyridine solution has adequate strength,pipet 10mLinto a 250-mLconical flask,add 25mLof pyridine and 50mLof water,and after about 15minutes add 0.5mLof a solution of phenolphthalein in pyridine (1in 100),then titrate with 0.5Nsodium hydroxide VS:it consumes between 37.6mLand 40.0mLof 0.5Nsodium hydroxide.

Procedure— Accurately weigh a suitable quantity,not exceeding 15g,of Poloxamer,calculated by multiplying the average molecular weight by 0.004,into a glass-stoppered,250-mLboiling flask.Carefully pipet 25mLofPhthalic anhydride–pyridine solution into the flask,touching the tip of the drained pipet to the protrusion in the flask.Add a few glass beads,and swirl to dissolve the specimen.Pipet 25mLofPhthalic anhydride–pyridine solution into a second,glass-stoppered,conical flask,add a few glass beads,and use as the reagent blank.(An additional 25-mLportion of pyridine may be added to both the test specimen and reagent blank,prior to refluxing,if necessary to ensure fluidity.)Heat both flasks,fitted with suitable reflux condensers,and allow to reflux for 1hour.Allow to cool,and pour two 10-mLportions of pyridine through each condenser.Remove the flasks from the condensers,add 10mLof water to each,insert the stoppers,swirl,and allow to stand for 10minutes.To each flask add 50.0mLof 0.66Nsodium hydroxide and 0.5mLof a 1in 100solution of phenolphthalein in pyridine.Titrate with 0.5Nsodium hydroxide VSto a light pink endpoint that persists for not less than 15seconds,recording the volume,in mL,consumed by the residual acid in the test solution asS,and that consumed by the blank as B.Calculate the average molecular weight taken by the formula: in which Wis the weight,in g,of the test specimen taken;and Nis the exact normality of the 0.5Nsodium hydroxide VS.

Solvent— Use deuterated water or deuterochloroform.

NMRreference— Use sodium 2,2-dimethyl-2-silapentane-5-sulfonate (for deuterated water)or tetramethylsilane (for deuterochloroform).

Test preparation— Dissolve 0.1g to 0.2g of Poloxamer in deuterated water containing 1%of sodium 2,2-dimethyl-2-silapentane-5-sulfonate to obtain 1mLof solution,or,if the Poloxamer does not dissolve in water,use deuterochloroform containing 1%of tetramethylsilane as the solvent.

Procedure— Transfer 0.5mLto 1.0mLof theTest preparation to a standard 5-mm NMRspinning tube,and if deuterochloroform is the solvent,add 1drop of deuterated water,and shake the tube.Proceed as directed forRelative Method of Quantitation under Nuclear Magnetic Resonance á761ñ,using the Test preparation volumes specified here,scanning the region from 0ppm to 5ppm,and using the calculation formulas specified here.Record as A1the average area of the doublet appearing at about 1.08ppm,representing the methyl groups of the oxypropylene units,and record as A2the average area of the composite band from 3.2ppm to 3.8ppm,due to the CH2Ogroups of both the oxyethylene and oxypropylene units and also the CHOgroups of the oxypropylene units,with reference to the sodium 2,2-dimethyl-2-silapentane-5-sulfonate or tetramethylsilane singlet at 0ppm.Calculate the percentage of oxyethylene,by weight,in the Poloxamer,taken by the formula: in which ais (A2/A1)–1.

Mercuric acetate solution— Place 50g of mercuric acetate in a 1000-mLvolumetric flask,and dissolve with about 900mLof methanol to which 0.5mLof glacial acetic acid has been added.Dilute with methanol to volume,and mix.Discard the solution if it is yellow.If it is turbid,filter it.Discard it if it is still turbid.Use fresh reagents if it is necessary to repeat the preparation of the solution.Protect the solution from light by storing it in an amber bottle in the dark.

Procedure— Transfer about 15.0g of Poloxamer to a 250-mLconical flask.Pipet 50mLof Mercuric acetate solution into the flask,and mix on a magnetic stirrer until solution is complete.Allow to stand for 30minutes with occasional swirling.Add 10g of sodium bromide crystals,and stir on a magnetic stirrer for about 2minutes.Without delay,add about 1mLof phenolphthalein TS,and titrate the liberated acetic acid with 0.1Nmethanolic potassium hydroxide VS.Perform a blank determination.Determine also the initial acidity as follows.Dissolve 15.0g of Poloxamer in 75mLof methanol that has been neutralized with methanolic potassium hydroxide to the phenolphthalein endpoint.Add about 1mLof phenolphthalein TS,and titrate with the same 0.1Nmethanolic potassium hydroxide VSunder a nitrogen sweep.Calculate the unsaturation,in mEq per g,taken by the formula: in which VU,VB,and VAare the volumes,in mL,of 0.1Nmethanolic potassium hydroxide used for titrating the test specimen,the blank,and the initial acidity,respectively;and Nis the normality of the titrant.

Stripped poloxamer— Place about 500g of Poloxamer 124into a suitable 3-neck,round-bottom flask equipped with a stirrer,a thermometer,a vacuum outlet,and a heating mantle.Evacuate the flask carefully at room temperature to a pressure of less than 10mm of mercury,applying the vacuum slowly to avoid excessive foaming due to entrapped gases.After any foaming has subsided,heat the flask to 80and continue to apply vacuum for 2hours;then cool to room temperature.Shut off the vacuum pump,and introduce nitrogen to bring the flask pressure back to atmospheric pressure.Transfer the Stripped poloxamer to a suitable nitrogen-filled container.

Standard preparation— [Caution—Ethylene oxide,propylene oxide,and 1,4-dioxane are toxic and flammable.Prepare these solutions in a well-ventilated fume hood. ]To a tared vial that can be sealed,add 50.0g ofStripped poloxamer.Add 60µLof 1,4-dioxane and 75µLof propylene oxide from a chilled syringe.Add ethylene oxide,using the following special handling procedure.Ethylene oxide—which is a gas at room temperature—is usually stored in a lecture-type gas cylinder or a small,metal pressure-bomb.Chill the cylinder in a refrigerator before use.Transfer about 5mLof the liquid ethylene oxide to a 100-mLbeaker chilled in wet ice.Using a gas-tight syringe that has been chilled in a refrigerator,transfer 15µLof the liquid ethylene oxide to the mixture.Immediately seal the vial,and shake on a vortex mixer for at least 30seconds.Transfer 0.20g of this solution to a tared vial that can be sealed,and addStripped poloxamer to obtain a Standard preparation having a final weight of 50.0g.Each gram of thisStandard preparation contains 1µg of ethylene oxide,5µg of propylene oxide,and 5µg of 1,4-dioxane.Transfer 1.00±0.01g of this solution to a 22-mLpressure headspace vial,and add about 0.01g of butylated hydroxytoluene.Seal with a silicone septum with or without a pressure-relief star spring and with a pressure-relief,aluminum,safety sealing-cap,and crimp the cap closed with a cap-sealing tool.

Test preparation— Transfer 1.00±0.01g of Poloxamer to a 22-mLpressure headspace vial,and add about 0.01g of butylated hydroxytoluene.Seal,cap,and crimp as directed for theStandard preparation.

Chromatographic system (see Chromatography á621ñ)— The gas chromatograph is equipped with a balanced-pressure automated headspace sampler,a flame-ionization detector,and a 0.32-mm ×50-m fused-silica capillary column coated with a 5-µm layer of stationary phase G27.The column temperature is programmed from 70(10-minute initial hold)to 240(10-minute final hold)at 10per minute,with the transfer line at 140and the detector and injection port at 250.The carrier gas is helium,flowing at a rate of about 1.6mLper minute.

Procedure— Separately place the vials containing theStandard preparation and theTest preparation in the automated sampler,and start the sequence so that the vial is heated at a temperature of 110for 30minutes before a suitable portion of its headspace is injected into the chromatograph.Set the automated sampler for a needle-withdrawal time of 0.3minute,a pressurization time of 1minute,an injection time of 0.08minute,and a vial pressure of 22psig with the vial vent off.Chromatograph theStandard preparation and theTest preparation,record the chromatograms,and measure the peak responses as directed forProcedure:the relative retention times are about 1.0,1.3,and 3.8,respectively,for ethylene oxide,propylene oxide,and 1,4-dioxane;and the resolution,R,between ethylene oxide and propylene oxide is not less than 2.0.Calculate the concentrations,in µg per g,of ethylene oxide,propylene oxide,and 1,4-dioxane in the portion of Poloxamer taken by the formula: in which Cis the concentration,in µg per g,of ethylene oxide,propylene oxide,or 1,4-dioxane in theStandard preparation;and rUand rSare the peak responses obtained from theTest preparation and theStandard preparation,respectively:not more than 1µg of ethylene oxide per g,not more than 5µg of propylene oxide per g,and not more than 5µg of 1,4-dioxane per g are found.