A:Infrared Absorption á197Kñ.

B: Prepare four Test solutionsof Dextran 40in water,in such a manner that the concentrations are accurately known and approximately evenly distributed in the range of 2%to 0.5%.Using a capillary tube viscosimeter having dimensions such that the flow time of water is not less than 100seconds,measure the flow times of water and of the Test solutionat 20.Calculate the viscosity numbers of each of the Test solutionsby the formula: in which RDis the ratio of the density of the individual Test solutionto that of water;tand t0are the flow times for the Test solutionand water,respectively;and Cis the concentration,in g per mL,of Dextran 40in the Test solution.Plot the viscosity numbers of each of the Test solutionsagainst their respective concentrations,and draw the straight line of best fit through the points and extrapolate to zero concentration:the value of the intercept is between 18and 23mLper g.

Test solution: 20mg per mL,heated,if necessary,on a water bath to dissolve.

Sulfate solution— To 1000mLof sulfuric acid add 5g of anhydrous cupric sulfate and 500g of potassium sulfate.Dissolve by heating,and store at 60.[note—If storage at 60is not possible,prepare a smaller quantity of Sulfate solution on the day of use,adjusting the proportions accordingly.]

Indicator— Dilute a mixture of 20mLof a 0.1%solution of bromocresol green in alcohol and 4mLof methyl red TSwith water to 100mL.

Procedure— Transfer 0.2g,accurately weighed,to a micro-Kjeldahl flask.Add 4mLof Sulfate solution.Heat until the solution exhibits a clear green color and the sides of the flask are free from carbonaceous material.Cool,and transfer the solution to a steam distillation unit.Rinse the Kjeldahl flask three times with 5mLof water,adding the washings to the solution.Add 15mLof 45%sodium hydroxide solution,immediately close the distillation apparatus,and commence steam distillation without delay.Receive the distillate in 1mLof Indicatorin a 100-mLflask,keeping the end of the condensing tube below the liquid surface for 5minutes and above the liquid surface for 1minute.Upon completion of the distillation,remove the receiving flask,and rinse the end of the condensing tube with a small quantity of water,adding the rinse to the distillate.Titrate the distillate with 0.010Nhydrochloric acid until the color changes from blue to reddish violet.Perform a blank determination,and make any necessary correction.The corrected volume of 0.010Nhydrochloric acid titrated does not exceed 0.14mL(0.01%,as N).

Test solution— Dissolve without heating 5.0g in 100mLof water,and distill the solution,collecting the first 45mLof the distillate.Dilute the distillate with water to 50.0mL,and mix.

Standard solution— To 25.0mLof the Test solutionadd 0.5mLof a 2.5%(w/v)solution of n-propyl alcohol.

Chromatographic system— The gas chromatograph is equipped with a flame-ionization detector and contains a 2-mm ×1.8-m column packed with support S3.The column temperature is maintained at about 160,the injection port temperature is maintained at about 240,and the detector is maintained at about 210.The carrier gas is nitrogen,flowing at a rate of about 25mLper minute.[note—Injector seals may deteriorate after multiple injections of the Standard and Test solutions.Inspect the seals before making a series of injections.]

Procedure— Separately inject equal volumes (about 1µL)of the Test solution,the Standard solution,and a 0.05%(w/v)solution of n-propyl alcohol and water,and measure the peak responses.After corrections for any impurities in the n-propyl alcohol solution and water,the total area of peaks from impurities in the Test solutiondoes not exceed the area of the n-propyl alcohol solution peak.

Mobile phase— Prepare a suitable degassed and filtered solution containing 7.1g of anhydrous sodium sulfate per Lin water.

Calibration solutions— Separately dissolve USP Dextran 4Calibration RS,USP Dextran 10Calibration RS,USP Dextran 40Calibration RS,USP Dextran 70Calibration RS,and USP Dextran 250Calibration RSin Mobile phaseto obtain solutions each containing 20mg per mL.

Marker solution— Prepare a solution in Mobile phasecontaining 3mg of dextrose and 3mg of USP Dextran VoMarker RSper mL.

System suitability solution— Prepare a solution of USP Dextran 40System Suitability RSin Mobile phasecontaining 20mg per mL.

Test solution— Prepare a solution of Dextran 40in Mobile phasecontaining 20mg per mL.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a refractive index detector and three 7.5-mm ×30-cm columns containing packing L38,and maintained at a constant temperature.Chromatograph the Marker solution,and record the peak responses as directed for Procedure:the elution profile shows two peaks,the first due to the Vomarker,the second due to dextrose.Determine the void volume,Vo,of the system as the inflection point of the ascending part of the first peak.Determine the total volume,VT,of the system as the maximum of the second peak;the tailing factor,t,of the dextrose peak is not more than 1.3;and the relative standard deviation of the ratio Vo/VTis not more than 1%.Chromatograph each of the Calibration solutionsseparately,and record the peak responses as directed for Procedure.Divide each profile into at least 60vertical sections of equal volume increments.(The actual number of sections is represented by the variable ain the equations below.)Record yi,the height above the baseline,corresponding to each value of vi,the volume eluted at that section.For each value of vi,calculate the distribution coefficient,Ki,by the formula: Find appropriate values of b1,b2,b3,b4,and b5,using a suitable method,*that,when substituted in the equation: and the resulting values ofMisubstituted,along with their corresponding values of yi,in the equation: give values of weight average molecular weight,bar(M)w ,within 5%of the labeled values for each of the Calibration solutionsand 180±2for dextrose.Chromatograph the System suitability solution,and record the peak responses as directed for Procedure.Calculate bar(M)wof the total molecular weight distribution using the same method as directed for the Calibration solutions,but inserting the now known values of b1,b2,b3,b4,and b5.It is between 39,000and 46,000.

Procedure— Chromatograph a 50-µLvolume of the Test solution,and record the peak responses.Calculate values of the weight average molecular weight,bar(M)w ,of the total molecular weight distribution of the high-fraction dextran,and of the low-fraction dextran as directed for System Suitabilityunder Chromatography á621ñthe values are between 35,000and 45,000,not more than 120,000,and not less than 5,000,respectively.With the values of b1,b2,b3,b4,and b5,obtained with the Calibration solutionsunder Chromatographic system,calculate the number average molecular weight,bar(M)n,of the total molecular weight distribution of the Test solutionby substituting the corresponding values of Mi,along with their corresponding values of yi,in the equation: The number average molecular weight,bar(M)n,is between 16,000and 30,000.Where Dextran 40is labeled as intended for use in the preparation of injectables,the ratio bar(M)w/bar(M)nis in the 1.4to 1.9range.