Principles and Practice of Chromatography - Chromatography Applications > Liquid Chromatography Applications > Page 80

Liquid Chromatography Applications

Liquid chromatography lends itself particularly to the separation of highly polar and large molecular weight materials that have very low volatility and, thus, can not be separated by gas chromatography. In addition, however, the technique is also used in trace analysis (e.g., drugs and drug metabolites in blood) where a concentration procedure is necessary and the sample is eventually regenerated in a small volume of liquid that can be injected directly onto an LC column. In general, the sample is passed through a short length of tube packed with an adsorbent which selectively removes the material of interest. The adsorbent is then washed and the adsorbed material extract with a small amount of suitable solvent and the solvent then inject onto the column.

An example of the use of this technique is in the determination of tetrahydrocannabinol carboxylic acid in urine. This substance appears in the urine of those subjects that have recently smoked marihuana. The tetrahydrocannabinol carboxylic acid can be extracted from the urine by means of a solid state extraction cartridge packed with a C18 reverse phase (a strongly dispersive packing containing bonded octyldecyldimethyl chains). The urine sample can be used direct, without pretreatment and the materials of interest are irreversibly adsorbed on the reverse phase solely by dispersive interactions. The actual procedure for extracting the tetrahydrocannabinol carboxylic acid according to Supelco is as a follows (1).

5 ml of urine was diluted with 5 ml of water and 0.5 ml of 10M potassium hydroxide and placed in a silanized glass tube. Note the tube with a non-polar wall was used to ensure that none of the acid was adsorbed on the hydroxyl groups of the glass. The tube was heated to 60oC for fifteen minutes and then cooled to room temperature. The sample was then adjusted to a pH of 4.5 with acetic acid. The C18 reversed-phase in the extraction tube was pre-conditioned with a mixture of 2 ml of methanol and 1 ml of 1% acetic acid. The hydrolyzed sample was allowed to percolate slowly through the tube by dropwise addition. The tube packing was then washed twice with 1 ml of a 20% aqueous solution of acetone, once with 1.5 ml aqueous 0.01M KH2PO4 , once with 0.5 ml of aqueous 0.01M Na2HPO4 and finally 0.5 ml of 1% aqueous acetic acid. The sample was then slowly desorbed from the reversed-phase with 1 ml of pure methanol and collected in a silanized glass tube. The sample was reduced in volume by evaporation in a stream of nitrogen and finally made up to a volume of 250ml with methanol.