Gel permeation chromatography cleanup system—

ELUANT: methylene chloride.

APPARATUS —The gel permeation chromatograph is equipped with a 25-mm ×100-cm column,packed with a slurry of styrene-divinylbenzene copolymer beads compressed to a bed length of approximately 77cm.The Eluantis pumped at a flow rate of about 4mLper minute.Set up the chromatograph,adjusting to discard the fraction eluting from 0to 43minutes,collect the fraction eluting from 43to 60minutes,and rinse for 20minutes,discarding the rinse fraction.

System suitability—

ELUTION OF LANOLIN ALCOHOLS —Melt a suitable quantity of USP Lanolin Alcohols RS,and pass through a fluted filter paper into a container.Transfer about 1.0g of warm filtered USP Lanolin Alcohols RS,accurately weighed,to a 10-mLvolumetric flask.Dilute with Eluantto volume,and mix.Transfer 5mLof this Standard solution to the gel permeation chromatographic column,and elute with Eluant.Collect 172to 240mLof the column effluent in a suitable evaporator.Evaporate the solvent,cool,weigh the evaporator,and calculate the amount of lanolin alcohols eluted in the evaporator.The column is suitable if not less than 99%of the lanolin alcohols elute in the first 172to 240mL.

Standard preparation— Dissolve a suitable quantity of USP Lanolin Alcohols RS,accurately weighed,in hexane,with the aid of warming if necessary,to obtain a solution having a known concentration of about 0.5mg per mL.[NOTE—Store this solution in the dark in a cold place for up to 4weeks.Before using,warm just sufficiently to dissolve any precipitate if necessary.]

Test preparation— Transfer 1g of Modified Lanolin,accurately weighed and previously melted to liquid form,by heating on a hot water bath if necessary,to a 10-mLvolumetric flask,dissolve in 7mLof Eluant,dilute with Eluantto volume,mix,and filter.Transfer 5.0mLof this solution to the column,and elute with 320mLof Eluant.Discard the first 172-mLfraction,collect the next 68-mLfraction (from 172to 240mL)in a suitable evaporator.Concentrate by evaporation on a steam bath to about 3mL,add about 50mLof hexane,and transfer this solution to a 100-mLvolumetric flask,adjusting the volume with hexane to 100mL.

Chromatographic system— The gas chromatograph is equipped with a flame-ionization detector maintained at 290,a 0.33-mm ×50-m fused silica capillary column bonded with a 0.50-µm layer of phase G2,a 0.32-mm ×50-cm fused silica uncoated guard column to protect the main analytical column.The column temperature is initially held at 210and programmed to rise to 280at the rate of 3per minute.Nitrogen is used as the carrier gas at a flow rate of about 7mLper minute,and is also used as the makeup gas at a flow rate of about 50mLper minute.

Procedure— Separately inject equal volumes (about 1µL)of the Standard preparationand the Test preparationinto the chromatograph,and allow both the Standard preparationand the Test preparationto elute for not less than 40minutes.Record the chromatograms,and measure the areas of all of the peaks.Calculate the quantity,in percentage,of free lanolin alcohols in the portion of Modified Lanolin taken by the formula: in which rUand rSare the total peak areas found in the Test preparationand the Standard preparation,respectively;Cis the concentration,in mg per mLof USP Lanolin Alcohols RSin the Standard preparation;Iis the volume,in mL,injected into the gel permeation chromatography column;Wis the weight,in g,of Modified Lanolin taken;and Kis the corrected fraction of free lanolin alcohols in the USP Lanolin Alcohols RSin the Standard preparationtaken by the formula: in which Aand Sare the acid value and saponification value,respectively,of USP Lanolin Alcohols RS:not more than 6%is found.