A: Infrared Absorption á197Kñ—To 1to 2mg each of USP Dextran 1RSand the sample add one to two drops of water,grind in an agate mortar for 1to 2minutes,add about 300mg of potassium bromide,and mix to a slurry.[NOTE—Do not grind.]Dry under vacuum at 40for 15minutes,and if it is not dry,continue drying for another 15minutes.Crush the residue,prepare a disk,and run the IRspectrum with a blank potassium bromide disk in the reference beam.

B: It meets the requirements of the test for Molecular weight distribution and average molecular weight.

Test solution— Proceed as directed forTest solutionin the test forLimit of alcohol and related impuritiesunderDextran 40,except to use 5.0g of Dextran 1.

Standard solution,Chromatographic system,and Procedure— Proceed as directed in the test forLimit of alcohol and related impurities underDextran 40.The total area of peaks from impurities in theTest solutiondoes not exceed the area of then-propyl alcohol solution peak.

Sulfate solution— To 1000mLsulfuric 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 solutionon the day of use,adjusting 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 Dextran 1,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,cautiously add 30mLof water,mix,and transfer the solution to a steam distillation unit.Rinse the Kjeldahl flask with three 5-mLportions of water,adding the washings to the solution.Add 15mLof 45%sodium hydroxide solution,immediately close the distillation apparatus,and start steam distillation immediately.Receive the distillate in 1mLofIndicatorand sufficient water to cover the end of the condensing tube.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 required to change the color does not exceed 0.15mL(110ppm of nitrogen).

Mobile phase— Prepare a filtered and degassed solution of sodium chloride containing 2.9g per liter.

Calibration solution— Prepare a solution containing about 0.45mg of isomaltotriose (3glucose units)and 0.60mg of sodium chloride per mL.

Reference solution— Prepare a solution of USP Dextran 1RSinMobile phasecontaining 6.0to 6.5mg per mL.

Test solution— Prepare a solution of Dextran 1inMobile phase containing 6.0to 6.5mg per mL.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a differential refractive index detector and two 10-mm ×30-cm columns in series that contain packing L54and are maintained at 20–25.The flow rate is 0.07to 0.08mLper minute,maintained constant to ±1%.

Procedure— Inject about 100µLof theCalibration solution,record the chromatogram,and note the retention times of the peaks.Separately inject equal volumes (about 100µL)of theReference solutionand Test solution,and record the chromatograms.Using the retention times in the chromatogram of Calibration solution,identify the peaks due to isomaltotriose and sodium chloride in the chromatograms of Reference solutionand Test solution.Disregard the peak due to sodium chloride in Reference solutionand Test solution.Calculate the weight-average molecular weight,MW,by the formula: in which Wiis the weight proportion of oligosaccharide i;andMiis the molecular weight of oligosaccharide i.Use the following molecular weight values for calculation: Calculate the amounts of the fractions with fewer than 3and with more than 9glucose units for the Reference solutionand theTest solution:theMwand amounts of the fractions obtained for theReference solutionare within the values stated in the data sheet that accompanies USP Dextran 1RS.TheMwof Dextran 1is between 850and 1150.The fraction with fewer than 3units of glucose is less than 15%,and the fraction with more than 9units of glucose is less than 20%.