Chromatographic tubes— Prepare three similar tubes,each about 260mm long and consisting of about 200mm of 25-mm tubing and about 6cm of 6-mm tubing.In each of the tubes,place a pledget of glass wool at a point where the 6-mm tubing is constricted slightly,about 2cm from the junction.

Citrate buffer— Mix equal volumes of 0.1Msodium citrate and 0.1Mcitric acid.

Standard preparation— Prepare a solution by dissolving an accurately weighed quantity of USP Morphine Sulfate RS,equivalent to about 40mg of anhydrous morphine,in 0.5mLof triethylamine contained in a 100-mLvolumetric flask,and add methanol to volume.Pipet 10mLof this solution into a 50-mLvolumetric flask,add 1mLeach of triethylamine and hydrochloric acid,and add water-saturated chloroform to volume.

Assay preparation— Evaporate 10.0mLof Paregoric (equivalent to about 4mg of morphine)on a steam bath under a stream of air to about 2mL,and cool.[NOTE—Avoid reducing the volume to less than 2mL.]Add 0.5mLof Citrate buffer.

Chromatographic columns— Fill the three tubes with adsorbent prepared as follows,using chromatographic siliceous earth as the base of the adsorbent,and tamp it firmly in place.Pack Column Iin two layers,the lower layer consisting of 3g of chromatographic siliceous earth mixed with 2mLof Citrate bufferand the upper layer of 3g of chromatographic siliceous earth mixed with the Assay preparation.Dry-rinse the beaker in which the components of the two layers have been mixed with 1g of chromatographic siliceous earth,and add it also to the top of Column I.Pack Column IIwith 3g of chromatographic siliceous earth mixed with 2mLof dibasic potassium phosphate solution (1in 5.75).Pack Column IIIwith 3g of chromatographic siliceous earth mixed with 2mLof sodium hydroxide solution (1in 50).Place a small pad of glass wool above each column packing.

Procedure— [NOTES—(1)Use water-saturated solvents throughout this procedure;(2)prepare eluants fresh daily;and (3)avoid bringing the solutions into contact with metal.]Wash Column Iwith 100mLof ether,followed by 100mLof chloroform,rinse the tip of the column with chloroform,and discard the solvents.In the following operations,rinse each column tip before discarding the column or changing receivers.Mount the three columns vertically so that the effluent from Column Iflows into Column II,and the effluent from the latter flows into Column III.Pass through the three columns 5mLof a 1in 5solution of triethylamine in chloroform,followed by four 10-mLportions of a 1in 100solution of triethylamine in chloroform,allowing each portion to pass through completely before subsequent additions.Discard Column I.Pass three 5-mLportions of the 1in 100solution of triethylamine in chloroform through the two remaining columns.Discard Column II.Wash Column IIIsuccessively with 10mLof the 1in 100solution of triethylamine in chloroform,50mLof chloroform,2mLof a 1in 10solution of glacial acetic acid in chloroform,and 50mLof a 1in 100solution of glacial acetic acid in chloroform.Discard all washings.Arrange to collect eluate from Column IIIin a 50-mLvolumetric flask containing 10mLof methanol and 1mLof hydrochloric acid.Elute the column with 5mLof a 1in 5solution of triethylamine in chloroform,followed by 33mLof a 1in 100solution of triethylamine in chloroform.Dilute with chloroform to volume,and mix.Concomitantly record the spectra of this solution and the Standard preparationin 1-cm cells,with a suitable spectrophotometer,from 255nm to 360nm,using chloroform as the blank,and plot the corresponding wavelength-absorbance curves.Correct the absorbance of each solution,at the wavelength of maximum absorbance at about 285nm,by extrapolating the portion of the base-line curve between 340nm and 310nm to this wavelength.Calculate the weight of anhydrous morphine,in mg per 100mLof Paregoric,taken by the formula: in which Wis the weight,in mg,of anhydrous morphine in the 50mLof Standard preparation,and AUand ASare the corrected absorbances of the solution from the Assay and the Standard preparation,respectively.